The significance of mass spectrometry lies in its unparalleled ability to accurately identify and quantify molecules in complex samples, providing invaluable insights into molecular structures and interactions. Here, we leverage diamond nanostructures as highly sensitive mass sensors by utilizing a self-excitation mechanism under an electron beam in a conventional scanning electron microscope (SEM). The diamond molecular balance (DMB) exhibits a practical mass resolution of 4.07 MDa, based on its notable mechanical quality factor and frequency stability, along with a broad dynamic range from MDa to TDa. This positions the DMB at the forefront of nanoelectromechanical system (NEMS)-based mass spectrometry operating at room temperature. Notably, the DMB demonstrated its ability to measure the mass of a single bacteriophage T4 by precisely locating the analyte on the device. These findings demonstrate the capability and potential of the DMB as a revolutionary tool for mass spectrometry at room temperature.