Biofilms are the source of numerous issues in the food, pharmaceutical, and production industries, making their control a major component of economic and public health. Among anti-biofilm strategies, enzyme-based products that target the biofilm matrix have proven effectiveness against multiple bacterial species. We tested the efficacy of a commercial product, Baso Bionil SL40^® (SL40; Stockmeier France, Saint-Jacques de la lande, France), against biofilms of Pseudomonas aeruginosa under various conditions of temperature, concentration, pH, and incubation time. SL40 contains two enzymes: a subtilisin protease and an α-amylase glycosidase. Our results showed that SL40 removed up to 85% of the biofilm biomass compared to tris solutions. SL40’s efficacy was strongly influenced by the presence of the enzymes and both temperature and concentration. Enzymatic activity was maintained from 20 °C to 60 °C and at pH values ranging from 5 to 9, with effective concentrations corresponding to SL40 dilutions from 3/200 to 1/200 in 50 mM tris solutions. Additionally, we observed that the P. aeruginosa biofilm biomass after pH 9 tris solution treatment was twice compared to a H₂O washing. Our results confirm the potential of enzymes against biofilms, highlight the need to define optimal application conditions, and support their integration into combined strategies for complete biofilm removal.