The aim of this study was to explore the antimicrobial activity of human follicular fluid (HFF), to test the hypothesis that different strains of the same bacterial species could display different patterns of susceptibility to antimicrobial action of HFF, and to preliminarily investigate the possible mechanism of antimicrobial action of this fluid. Antimicrobial activity of 60 samples of HFF toward 30 Streptococcus agalactiae strains was determined by the agar diffusion method and broth dilution method. To explore the mechanism of antimicrobial activity, biochemical analyses were performed with selected fluid samples. The obtained results indicate that 38.3% fluid samples did not inhibit bacterial growth, 53.3% showed moderate and 8.3% high antimicrobial activity. The tested effect of HFF on S. agalactiae strains was bactericidal and was not strain dependent. Lysozyme activity was detected in HFF exhibiting antimicrobial activity. There were no statistically significant differences in concentrations of estradiol, progesterone, transferrin, iron, total protein and albumin levels among tested samples regardless of the different rate of antimicrobial activity. The obtained results indicate that lysozyme is most probably a crucial antibacterial agent in this fluid; however, some other still unidentified factors may contribute to it.