The flavonoid fraction isolated from the ethyl acetate fraction (BF-1) of Butea frondosa (L.) stem bark exhibited distinct antimicrobial activity when tested against 129 bacterial strains belonging to 9 different genera of both gram-positive and gram-negative types. Minimum inhibitory concentration (MIC) of the fraction BF-1 was determined following NCCLS guidelines using the agar dilution method. Twenty-four out of 36 strains of Staphylococcus aureus were inhibited by 50-200 mg/l of the fraction. This fraction also inhibited strains of Bacillus spp., Shigella spp., Salmonella spp. and even a few Pseudomonas at concentrations between 50-200 mg/l. Other bacteria including Escherichia coli, Vibrio cholerae and V. parahaemolyticus were moderately sensitive to BF-1. In the in vivo studies, this fraction offered significant protection to Swiss albino mice at a concentration of 80 microg/mouse (p<0.001) when they were challenged with 50 median lethal dose of Salmonella enteritidis NCTC 74. A fraction named BF-1 that was isolated from an ethyl acetate fraction of Butea frondosa provided protection against an infection from a Salmonella enteritidis NCTC strain.