Current cleaning and sanitation protocols may be ineffective in cleaning separation membranes and result in the formation of resilient multispecies biofilms. These old biofilms may show bacterial predominance on prolonged use of the membranes. In our previous study, we isolated organisms such as Bacillus subtilis, Bacillus licheniformis, Exiguobacterium aurantiacum, and Acinetobacter radioresistens from an 18-mo old reverse osmosis membrane. Competitive exclusion studies revealed the predominance of B. subtilis within the membrane biofilm microflora. This study investigates the antimicrobial activity of B. subtilis as a cause of its predominance. The culture was incubated in tryptic soy broth (TSB) at 37°C, and microfiltered to prepare cell-free extracts (CFEs) at 8-, 10-, 12-, 14-, 16- and 18-h intervals. The CFEs were freeze-dried and re-suspended in minimum quantities of HPLC grade water to create concentrated solutions. The antimicrobial activities of CFEs were tested using agar-well assay against the biofilm constitutive microflora and some common food pathogens. The experiments were conducted in triplicates and means were compared for significant differences using a general linear mixed model procedure of SAS. The results indicated the highest antimicrobial activity of 12 h CFE of B. subtilis against other Exiguobacterium sp., E. auranticum, A. radioresistens, Listeria monocytogenes, and Bacillus cereus, with average inhibition zone sizes of 16.5, 16.25, 20.6, 18.0, and 13.8 mm, respectively. On treating with proteinase K, the CFE completely lost its antimicrobial activity, establishing it to be a proteinaceous compound. The amino acid profiling revealed the total crude protein in CFE to be 51% (wt/wt) having its major constituent to be glutamic acid (13% wt/wt). The CFE was thermally stable on exposure to the common temperature used for sanitizer applications (23.8°C for 5 and 10 min). Based on this study, the proteinaceous antimicrobial compound produced by B. subtilis may result in its predominance.