The goal of this study was to investigate the effect of antibiotic sequence on combination regimens against Pseudomonas aeruginosa in an in vitro infection model. Ceftazidime plus ciprofloxacin and ceftazidime plus tobramycin were dosed every 12 h for 48 h using simultaneous or staggered administration. Simultaneous dosing and ceftazidime followed by ciprofloxacin or tobramycin were significantly more active at both 24 h (p = 0.03) and 48 h (p < 0.0001) than ciprofloxacin or tobramycin followed by ceftazidime. Final bacterial kill was sixfold greater with the former regimens. This study showed that antibiotic sequence had a significant and class dependent effect on antibacterial response. The clinical relevance of these observations warrants further investigations in animal models.