This study evaluated antimicrobial properties of synovial fluid against 3 gram-positive organisms: Staphylococcus aureus, Streptococcus pyogenes, and Staphylococcus epidermidis. Synovial fluid was collected from the knees of 52 patients. Three gram-positive bacteria culture lines were incubated in wells containing either synovial fluid and broth, or broth alone. Fluid was plated and incubated, and colonies were counted after 1, 4, and 24 hours to determine bacterial growth. Statistically significant differences in bacterial counts were found between control and experimental groups at 0, 4, and 24 hours for all 3 bacterial species. Bacterial counts in the control specimens demonstrated exponential growth over 24 hours as would be expected in the absence of growth inhibition. In contrast, bacterial counts in all of the synovial fluid specimens decreased steadily over 24 hours. These results demonstrate synovial fluid possesses potent bactericidal activity against the most common gram-positive pathogens responsible for septic arthritis. Delineating these antimicrobial properties further may offer avenues to augment the body's protection against bacterial infections.