Spores can resist pasteurization, germinate, and grow in the dairy products during storage, causing spoilage. The aim of this work was to investigate the antimicrobial properties of several catalase preparations (Cat) against spore-forming bacteria isolated from dairy sources and their possible applications in milk and cleaning of separation membranes. The antimicrobial activity of a food-grade (FG) commercially available Cat produced by Aspergillus niger, a non-food-grade Cat (NFG), and a Cat from bovine liver (BL) against Bacillus sporothermodurans (Bs), Geobacillus stearothermophilus (Gs), Bacillus mojavensis (Bc), Bacillus licheniformis (K1), and Bacillus spp. (10/1) was assessed by the agar diffusion assay and broth microdilution. The effect of FG Cat on biofilms formed by 2 single strains of slime-producing Bacillus (Bc and K1) and a cocktail of 4 strains (Bc, Bs, Gs and 10/1) was studied alone (150 mg/mL) and after a pretreatment with 0.1% Tween 20-PBS buffer. Last, a cocktail of 10³ spores/mL of 4 strains of Bacillus (Bc, Gs, Bs, and 10/1) inoculated in UHT milk was challenged with 0, 6.25 and 12.5 mg/mL of FG Cat for 30 min and the number of survivors was determined. The FG and NFG Cat inhibited the growth of vegetative cells of all tested species of Bacillus at 12.5 mg/mL with inhibition zones of up to 25 mm, while BL did not show any inhibition. The FG Cat reduced the number of viable cells in one-day-old Bc and K1 biofilms by 2 and 4.87 log cfu/cm² respectively (P < 0.05). While the application to a 3-d-old multispecies biofilm reduced the viable counts by only 0.73 log cfu/cm², a pretreatment with Tween 20 followed by FG, resulted in a 1.18 log cfu/cm² reduction (P < 0.05). The FG Cat at 12.5 mg/mL reduced the number of germinating spores in milk by 36.7%. The FG Cat lost antimicrobial activity after heating at 100°C for 10 min. This works describes for the first time the antimicrobial activity of Cat preparations against bacterial spores, which would create new opportunities for the dairy industry to control germination and outgrowth of spore-forming bacteria (patent pending).