Calls for his testimony come amid claims that the adviser directly linked Mr Trump to a plan to freeze aid to Ukraine in exchange for a political favour.
The claims, reportedly made in a book manuscript, are central to the trial that has resumed on Monday.
Mr Trump denies the charges.
But the claims allegedly made by Mr Bolton could damage the president’s defence.
The president is accused of withholding military aid to pressure Ukrainian President Volodymyr Zelensky to start a corruption investigation into Mr Biden, and his son Hunter.
Mr Trump told reporters last week that he did not want Mr Bolton to testify, citing national security concerns.