Microspheres or droplets are increasingly finding various biomedical applications as drug microspheres and multicellular spheroids. Single nozzle-based continuous jetting with the help of acoustic excitation and/or carrier stream is a basic process for monodisperse microsphere fabrication. Precise control of microsphere size and size distribution in single nozzle jetting is still of great manufacturing interest. The objective of this study is to numerically model a glycerol-water microsphere fabrication process during acoustic excitation-based single nozzle continuous jetting. Using a volume of fluid method, this study has investigated the effects of material properties and fabrication conditions such as the acoustic excitation frequency and amplitude and the carrier stream velocity on the size of microspheres fabricated. (1) The microsphere diameter decreases as the glycerol volume percentage increases. (2) The excitation frequency and pressure have a pronounced effect on the microsphere size. The microsphere diameter decreases as the excitation frequency increases, and the microsphere diameter increases with the excitation pressure amplitude. (3) The microsphere size decreases as the carrier stream velocity increases.