In this study, the drop-on-demand patterning of bacterial cells on a raw silicon wafer was newly conducted with an electrospray pulsed jet. We produced various sized line patterns and spot patterns of bacterial cells on the silicon wafer under varying experimental conditions of frequency, flow rate, and translational speed of die electrospray system in one and two-dimensional ways. Especially, the electrospray's pulsed jet of bacterial solution produced regular patterns of around 10 pm diameter spots, each of which contained a single bacterial cell. By enabling drop-on-demand patterning and manipulation of single bacterial cells on a raw wafer in the gas phase, this new technique broadens the scope of biological experiments beyond liquid phase treatments to solid and gas-phase treatments.