Enzymatic cleaners are generally used during cleaning in place (CIP) processes to improve the cleanability of dairy separation membranes. Many of the commercial enzymatic cleaners, however, contain general action enzymes, not specifically designed to degrade recalcitrant biofilm matrices. In our previous screening, some enzymes showed a greater biofilm removal on reverse osmosis (RO) membranes, as compared with commercial enzyme-based cleaners. This project aims to evaluate the efficacy of a protease (EC 3.4.24.31, named S1), an alkaline phosphatase (EC 3.1.3.1, S2), and a lactase (EC 3.2.1.23, S3) in removing biofilms on diverse dairy separation membranes, for RO (KMS HRX: TFC polyamide), and Ultrafiltration (UF) processes (HFK-131: polyethersulfone, and HFM-180: polyvinylidene difluoride) (Koch membrane systems, Wilmington, MA). Forty-eight-hour-old mixed species biofilms, constituting common dairy sporeformers Bacillus licheniformis, B. coagulans, B. sporothermodurans, and Geobacillus stearothermophilus, were developed on the respective membranes (4cm²) under lab conditions. Tryptic soy broth at 37°C served as the immersion medium. All enzymes and buffer solutions used were prepared following manufacturer recommendations (Sigma-Aldrich, Saint Louis, MO). Membranes in triplicates were rinsed with sterile distilled water, followed by separately cleaning for 45 min at 55°C with individual enzyme solutions at 0.2 U/mL (S1), 0.1 DEA/mL (S2), and 0.01 U/mL (S3). All assays were repeated 3 times, and data were statistically analyzed. The residual viable cell numbers were estimated by swabbing, and plating on plate count agar. Percentage reductions in viable counts for S1, S2 and S3, respectively, were 99.93, 99.69 and 90.74% cfu/cm² for biofilms formed on RO, 99.99, 99.91 and 99.40% for UF HFK-131, and 98.23, 0.0 and 48.0% for UF FM-180. In conclusion, S1 was the most effective enzyme for reducing multispecies biofilms on all membrane types. Additionally, the most resistant biofilms were observed on HFM-180. These findings suggest that for better cleaning of any membrane material, it will be critical to design a specific enzyme-based formulation, depending on a particular biofilm matrix.