Microbial attachment and colonization on separation membranes lead to biofilm formation. The constitutive microflora might compete and result in certain species emerging as predominant, especially within older biofilms. To understand the microbial interactions within biofilms, the emergence of predominance was studied in the current investigation. An 18-mo-old reverse osmosis membrane was procured from a whey processing plant. The membrane pieces (1 × 1 inch²) were neutralized by dipping in Latheen broth. Their resuscitation was done in Tryptic Soy Broth (TSB) at 37°C, followed by plating on Tryptic Soy Agar (TSA) to recover the constitutive microflora. Distinct colonies of isolates were further identified using MALDI-TOF as Bacillus subtilis, Bacillus licheniformis, Exiguobacterium aurantiacum, Acinetobacter radioresistens, and 2 unidentified species of Exiguobacterium and Bacillus. Further, the competitive exclusion study helped to establish the predominance using a co-culturing technique. Fifteen combinations of 2 isolates each were prepared. For which, isolates were spiked in the ratio of 1:1 in TSB, incubated at 37°C for 24 h, followed by plating on TSA. The isolates on plates were distinguished based on colony morphology, Gram staining, and MALDI-TOF. For establishing the predominance, plate counts were compared using ANOVA. In all the coculture combinations, B. subtilis emerged as predominant with a mean log counts of 6.73 ± 0.23 cfu/ mL. It was interesting to note that another isolate, B. licheniformis, competed equally with B. subtilis, while not with others. The predominance of B. subtilis was further validated using the process of natural selection, where the broth with overnight-incubated membrane piece (with mixed species biofilm) was inoculated in fresh TSB and incubated for another cycle. Five such sequential transfers resulted in demonstrating the predominance of B. subtilis based on its population density.