The objective of this study was to assess the biofouling characteristics of the Bacillus biofilm formed on reverse osmosis (RO) membranes. For the study, a sporogenic Bacillus sp. was isolated from the seawater intake to a RO process, with two distinct sets of experiments performed to grow the Bacillus biofilm on the RO membrane using a lab-scale crossflow membrane test unit. Two operational feds were used, 9 L sterile-filtered seawater and 109 Bacillus cells, with flow rates of 1 L/min, and a constant 800 psi-pressure and pH 7.6. From the results, the membrane with more fouling, in which the observed permeate flux decreased to 33% of its initial value, showed about 10 and 100 times greater extracellular polymeric substances and spoOA genes expressions, respectively, than the those of the less fouled membrane (flux declined to 20% of its initial value). Interestingly; however, the number of culturable Bacillus sp. in the more fouled membrane was about 10 times less than that of the less fouled membrane. This indicated that while the number of Bacillus had less relevance with respect to the extent of biofouling, the activation of the genes of interest, which is initiative of biofilm development, had a more positive effect on biofouling than the mass of an individual Bacillus bacterium.