New approaches to antimicrobial therapy for ocular pathogens must overcome organisms that are resistant to current therapeutic modalities. This investigation examined the antimicrobial activity of novel antimicrobial neutrophil peptides (defensins NP-1 and NP-5) against isolates from clinical ocular microbial infections in humans and horses. The test panel of human clinical isolates included Candida albicans, an alpha-hemolytic Streptococcus, Streptococcus pneumoniae, Pseudomonas aeruginosa, and Morganella morganii. The test panel of equine pathogens included three clinical isolates of P aeruginosa and two clinical isolates of Staphylococcus aureus. The equine isolates were chosen for their relative resistance to commonly employed antimicrobial therapy. The two defensins differed markedly in their bactericidal activity. Defensin NP-5, at a 50-micrograms/mL concentration, exhibited minimal bactericidal activity against the majority of isolates of the test panel. The inferior microbicidal activity of NP-5 is consistent with previously published results. However, at this concentration, NP-5 did exhibit appreciable bacteriostatic activity against human ocular pathogens M morganii (74%), alpha-hemolytic Streptococcus (57%), and P aeruginosa (93%) during the 2-hour incubation period. In contrast, defensin NP-1 at 10 micrograms/mL exerted potent microbicidal activity against all isolates, effecting a 2 to 3 log10 decrease in colony-forming units within a 60-minute incubation period. Under the assay conditions employed, these findings demonstrate: (1) two distinct mechanisms by which defensins exert their antimicrobial activity against microbial pathogens associated with clinical ocular disease in humans and horses, and (2) that rabbit defensin NP-1 is a potent antimicrobial agent against a wide array of ocular pathogens.