The in vitro activity of the dual-action antibacterial agent Ro 23-9424 was compared with those of cefotaxime, ceftazidime, ciprofloxacin, fleroxacin, imipenem, and amikacin against 358 aerobes and anaerobes. The MIC ranges, MICs for 50 and 90% of the strains (MIC50s and MIC90s), and percentage of strains susceptible for each agent at the recommended susceptible MIC breakpoint were determined for each genus. The MIC90s (micrograms per milliliter) of the agents against members of the family Enterobacteriaceae were as follows: ciprofloxacin, 0.063; Ro 23-9424, fleroxacin, and imipenem, 0.5; ceftazidime, 2; amikacin, 4; and cefotaxime, 16. The MIC90s (micrograms per milliliter) against Pseudomonas and Acinetobacter spp. were as follows: ciprofloxacin, 2; ceftazidime and imipenem, 8; Ro 23-9424, 16; fleroxacin, 32; amikacin, 64; and cefotaxime, 128. Against gram-positive bacteria, excluding the enterococci, the MIC90s (micrograms per milliliter) were as follows: ciprofloxacin, 1; imipenem, 4; Ro 23-9424 and fleroxacin, 8; amikacin, 64; and ceftazidime and cefotaxime, greater than 128. Against gram-positive bacteria, including the enterococci, the MIC90s changed only for the following agents: Ro 23-9424, 16 micrograms/ml; and amikacin, 128 micrograms/ml. Strains of Branhamella catarrhalis, Haemophilus influenzae, and Neisseria gonorrhoeae were 100% susceptible to Ro 23-9424, cefotaxime, ciprofloxacin, and fleroxacin, while the other three agents showed somewhat less activity only against N. gonorrhoeae. Against anaerobes, imipenem was the most effective agent, while the activities of the other six agents were variable.