BAL9141, the active principle of prodrug BAL5788, is a parenteral cephalosporin active against oxacillin-resistant Staphylococcus aureus. Previous studies have reported the cidal action of BAL9141 towards limited numbers of isolates by time-kill methods. We tested >100 staphylococci, streptococci, and Gram-negative isolates by broth microdilution methods to determine the antibacterial activity of BAL9141 and to compare minimum inhibitory concentrations (MICs) with minimum bactericidal concentrations (MBCs). Among the 53 staphylococcal isolates, all but one (98%) had a, MBC:MIC ratio =<2. Forty-five percent (five strains) of the penicillin-nonsusceptible Streptococcus pneumoniae isolates had an MBC:MIC ratio >4. Among 22 Enterobacteriaceae, 73% of isolates had MBC:MIC ratios of 2 or less; three strains with ratios greater than 4 were Serratia marcescens. BAL9141 displayed a predominant bactericidal activity against the majority of Gram-positive and -negative species, including resistant subsets, making it an attractive candidate for further development.