Lactose oxidase (LO) oxidizes lactose to lactobionic acid, producing H2O2 that could act as an antimicrobial and activate the lactoperoxidase system (LPS), an antimicrobial system present in milk. We evaluated the antimicrobial effect of LO alone and with the components of the LPS: NaSCN and lactoperoxidase. An increase in LO caused higher reductions of Pseudomonas fragi in pasteurized milk. A total bacterial growth curve in raw milk showed a longer lag phase and lower counts when the LPS was activated by LO compared to the control. The antimicrobial effect of LO against foodborne pathogens and spoilage microorganisms was evaluated through an overlay inhibition assay. LO was applied in different media to evaluate the effect of lactose and SCN-. All the indicators were inhibited by LO in presence of lactose. However, NaSCN had no effect on the inhibition. The antimicrobial effect was attributed to H2O2 produced from the oxidation of lactose.