Bovine mastitis is the most significant economic drain on the worldwide dairy industry. Concerns regarding poor cure rates, emergence of bacterial resistance, and residues in milk necessitate development of alternative therapeutic approaches to antibiotics for treatment of mastitis. A variety of free fatty acids and their monoglycerides have been reported to exert antimicrobial activity against a wide range of microorganisms. The objective of our study was to examine the efficacy of caprylic acid, a short-chain fatty acid, and its monoglyceride, monocaprylin, to inactivate common mastitis pathogens, including Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis, Staphylococcus aureus, and Escherichia coli. Milk samples containing 50 mM or 100 mM caprylic acid, and 25 mM or 50 mM monocaprylin were inoculated separately with a 3-isolate mixture of each of the 5 pathogens, and incubated at 39 degrees C. Populations of surviving bacteria were determined at 0 min, 1 min, 6 h, 12 h, and 24 h of incubation. Both caprylic acid and monocaprylin reduced all 5 pathogens by >5.0 log cfu/mL after 6 h of incubation. Among the bacterial species tested, Strep. agalactiae, Strep. dysgalactiae, and Strep. uberis were most sensitive, and E. coli was most tolerant to caprylic acid and monocaprylin. Results of this study indicate that caprylic acid and monocaprylin should be evaluated as alternatives or adjuncts to antibiotics as intra-mammary infusion to treat bovine mastitis.