Many companies in the food industry apply reverse osmosis (RO) membranes to ensure high-quality reuse of water. Biofouling is however, a common, recalcitrant and recurring problem that blocks transport over membranes and decreases the water recovery. Microorganisms adhering to membranes may form biofilm and produce an extracellular matrix, which protects against external stress and ensures continuous attachment. Thus, various agents are tested for their ability to degrade and disperse biofilms. Here, we identified industrially relevant bacterial model communities that form biofilms on RO membranes used for treating process water before reuse. There was a marked difference in the biofilm forming capabilities of bacteria isolated from contaminated RO membranes. One species, Raoultella ornithinolytica, was particularly capable of forming biofilm and was included in most communities. The potential of different enzymes (Trypsin-EDTA, Proteinase K, α-Amylase, β-Mannosidase and Alginate lyase) as biofouling dispersing agents was evaluated at different concentrations (0.05 U/ml and 1.28 U/ml). Among the tested enzymes, β-Mannosidase was the only enzyme able to reduce biofilm formation significantly within 4 h of exposure at 25 °C (0.284 log reduction), and only at the high concentration. Longer exposure duration, however, resulted in significant biofilm reduction by all enzymes tested (0.459–0.717 log reduction) at both low and high concentrations. Using confocal laser scanning microscopy, we quantified the biovolume on RO membranes after treatment with two different enzyme mixtures. The application of proteinase K and β-Mannosidase significantly reduced the amount of attached biomass (43% reduction), and the combination of all five enzymes showed even stronger reducing effect (71% reduction). Overall, this study demonstrates a potential treatment strategy, using matrix-degrading enzymes for biofouled RO membranes in food processing water treatment streams. Future studies on optimization of buffer systems, temperature and other factors could facilitate cleaning operations based on enzymatic treatment extending the lifespan of membranes with a continuous flux.