The most common antimicrobial polypeptides in breast milk are lactoferrin (LF), lactalbumin (LA), lysozyme (LC), and lactoperoxidase (LP). The aim of the present study was to evaluate the combined effect of these polypeptides on the cells of Candida albicans, Staphylococcus aureus, and Escherichia coli in vitro by spectrophotometric method. The antimicrobial effect of LP on the microbial cells was tested without connection with the lactoperoxidase system. It was shown that LA alone did not demonstrate any antimicrobial activity. However, the LF, LP, and LC in the concentration range from 2.5 mg/ml to 20 mg/ml exhibited a direct microbicidal effect in a significantly dose-dependent manner. The combined effect of the preparations was studied at a concentration of 5 mg/ml. The total antimicrobial activity of the combination of LF and LC on S. aureus and E. coli was significantly lower than the sum of the activities of individual preparations, i.e., there was an antagonistic effect, whereas there was a slight synergy towards C. albicans. For the LF and LP pairs, the experimental and calculated values ​of the total activity turned out to be almost the same against both types of bacteria, whereas in the case of yeast, there was a significant antagonistic effect. For the pair LP and LC in both species of bacteria, there was an antagonistic effect, and in relation to C. albicans, these results were practically the same. Thus, the combined actions of all the three antimicrobial peptides have shown a prominent antagonistic effect.