Mechanisms for protecting spermatozoa, and the testes that produce them, from infection are essential, given the importance of these cells and organs for the fertility of the individual and perpetuation of the species. This is borne out by the publication of numerous papers on this subject over the last 50 years. We extended our work and that of others on the anti-infectious defense system of the male genital tract, using a new strategy for the direct identification of antibacterial molecules in human seminal plasma. We subjected a liquefied seminal plasma cationic fraction to reversed-phase HPLC, monitored microbicidal activity by gel overlay and radial diffusion assays, and identified the proteins and/or peptides present in each active fraction by mass spectrometry. In addition to proteins with known potent microbicidal activity—phospholipase A2, lactoferrin, and lysozyme—we also found that peptides produced by cleavage of semenogelin I, the predominant human semen coagulum protein, had high levels of antibacterial activity.