State prosecutors in Manhattan who are investigating former President Donald J. Trump and his family business are sharpening their focus on the company’s long-serving chief financial officer, asking witnesses questions about his dealings at the company, according to people with knowledge of the matter.

The increased focus on the executive, Allen H. Weisselberg, could step up pressure on him to cooperate with the investigation if the prosecutors unearth evidence of wrongdoing on his part. He has served as the Trump Organization’s financial gatekeeper for more than two decades and could be a vital source of information for the government about the inner workings of the company.

In recent weeks, the prosecutors working for the Manhattan district attorney, Cyrus R. Vance Jr., have been interviewing witnesses who know Mr. Weisselberg and have asked at least one witness about Mr. Weisselberg’s sons, Barry and Jack Weisselberg, according to two of the people with knowledge of the matter.

Barry Weisselberg has been the property manager of Trump Wollman Rink in Central Park, and Jack works at Ladder Capital, one of Mr. Trump’s biggest lenders.

The district attorney’s office has not accused Mr. Weisselberg or his sons of any wrongdoing, and there is no indication that the sons are a focus of the investigation.

If the prosecutors were to secure Allen Weisselberg’s cooperation, it might provide a significant boost to the long-running investigation and deliver a blow to Mr. Trump, who has long depended on Mr. Weisselberg’s unflinching loyalty.

Prosecutors are examining, among other things, whether Mr. Trump and the Trump Organization falsely manipulated property values to obtain loans and tax benefits. For his part, Mr. Trump, a Republican, has repeatedly dismissed the inquiry as a politically motivated “witch hunt” and “fishing expedition” by Mr. Vance, a Democrat.

Mr. Weisselberg, 73, an accountant, began his career working for Mr. Trump’s father and has overseen the Trump Organization’s books for decades. He recently ran the business with Mr. Trump’s adult sons during the Trump presidency and remained loyal to the company even after his name surfaced during congressional and federal investigations into Mr. Trump or his business.

More recently, Mr. Weisselberg has become a figure of interest to the district attorney’s investigation, according to the people with knowledge of the matter. In addition to potential financial crimes, the investigation has focused on the possible role Mr. Weisselberg and other Trump Organization employees played in hush money payments made during the 2016 presidential campaign to two women who said they had affairs with Mr. Trump.

A spokesman for Mr. Vance declined to comment on the investigation. A lawyer for Allen Weisselberg, Mary E. Mulligan, also declined to comment, as did a representative of the Trump Organization.

Last month, Mr. Vance’s team won a big victory when the Supreme Court rejected a last-ditch attempt by Mr. Trump to block a subpoena for his financial records, ending the former president’s nearly 18-month battle to stop investigators from seeing his tax returns.

“For more than two years, New York City has been looking at almost every transaction I’ve ever done, including seeking tax returns which were done by among the biggest and most prestigious law and accounting firms in the U.S.,” Mr. Trump said after the Supreme Court ruled.

While awaiting the ruling, the prosecutors have broadened the inquiry to include an array of dealings involving some of Mr. Trump’s most prominent properties — including Trump Tower on Fifth Avenue in Manhattan, various Trump hotels and the Seven Springs estate in Westchester County.

The prosecutors are examining whether the Trump Organization inflated the value of some of his properties to obtain the best possible loans while lowballing the values to reduce property taxes, people with knowledge of the matter have said. The prosecutors are also investigating the Trump Organization’s statements to insurance companies about the value of various assets.

The prosecutors have subpoenaed Mr. Trump’s two main lenders, Deutsche Bank and Ladder Capital, which are cooperating with the investigation. Ladder Capital sold its Trump Organization loans, along with others, to investors years ago and thus no longer owns them.

The Trump Organization’s lawyers are expected to argue to prosecutors that the company could not have duped sophisticated financial institutions who provided loans because they did their own analysis of Mr. Trump’s properties without heeding the company’s assessments. The company’s lawyers are also likely to emphasize that the practice of providing different valuations for the same property depending on the situation is widespread in New York’s real estate industry.

When Mr. Vance’s office opened its investigation more than two years ago, it began with an examination of the hush money payments. In particular, the prosecutors scrutinized how the company accounted for $130,000 it gave Michael D. Cohen, Mr. Trump’s former fixer, as reimbursement for money he paid to buy the silence of one of the women, Stormy Daniels, a pornographic film actress who said she had an affair with Mr. Trump.

Mr. Cohen pleaded guilty to federal campaign finance charges for his role in arranging the payments. Now he is cooperating with Mr. Vance’s prosecutors, who have interviewed him several times, according to people with knowledge of the meetings. Lanny Davis, a lawyer for Mr. Cohen, declined to comment.

Testifying before Congress two years ago, Mr. Cohen pinned blame for the hush-money scheme on Mr. Weisselberg, who he said helped devise a strategy to mask the Trump Organization’s reimbursements to Mr. Cohen for his payment to Ms Daniels. The federal prosecutors who charged Mr. Cohen did not accuse Mr. Weisselberg of wrongdoing.

Susan C. Beachy contributed research.