Initial deposition of bacteria is a critical stage during biofilm formation and biofouling development in membrane systems used in the water industry. However, the effects of hydrodynamic conditions on spatiotemporal deposition patterns of bacteria during the initial stages of biofilm formation remain unclear. Large field epifluorescence microscopy enabled in situ and real-time tracking of Bacillus subtilis in a forward osmosis system with spacers during the first 4 h of biofilm formation. This study quantitatively compares the spatiotemporal deposition patterns between different hydrodynamic conditions: high and low permeate water flux (6 or 30 L m-2 h-1) as well as high and low crossflow velocity (1 or 14 cm s-1). Low crossflow velocity and high permeate water flux maximized bacterial attachment to the membrane surface, which was 60 times greater (6 × 103 cells mm-2) than at high crossflow velocity and low permeate water flux (<100 cells mm-2). Imaging at 30 s intervals revealed three phases (i.e., lag, exponential, and linear) in the development of deposition over time. Quantification of spatial deposition patterns showed that an increase in the ratio of permeate water flux to crossflow velocity led to a homogeneous deposition, while a decrease had the opposite effect. The insights of this research indicate that an appropriate choice of hydrodynamic conditions can minimize bacteria accumulation prior to biofilm formation in new and cleaned FO membrane systems treating water of high fouling propensity.