Pseudomonas aeruginosa, the predominant causative pathogen of chronic lung infection in patients with cystic fibrosis, may grow under anaerobic conditions as biofilms in the lungs of cystic fibrosis patients. To determine if growth under anaerobic conditions affects the antimicrobial susceptibility of P. aeruginosa, the susceptibility of clinical isolates of P. aeruginosa grown planktonically and as biofilms to a range of antibiotics was determined. Growth under anaerobic conditions did not reduce the ability of ceftazidime, meropenem, aztreonam, piperacillin, or piperacillin/tazobactam to inhibit planktonic growth, with MIC50 values for these antibiotics remaining unchanged or decreasing. However, tobramycin was less effective at inhibiting planktonic bacterial growth under anaerobic conditions, with the MIC50 of tobramycin increasing twofold. Growth under anaerobic conditions also decreased the bactericidal activity of tobramycin, with the MBC50 of tobramycin increasing fourfold. The killing kinetics of tobramycin was also examined under aerobic and anaerobic conditions for selected isolates. When isolates 6A and 12A were grown aerobically, concentration-dependent decreases in total viable count were apparent with tobramycin. In contrast, when these isolates were grown anaerobically, tobramycin at the same concentrations did not decrease the total viable count. When isolates were grown as biofilms under both aerobic and anaerobic conditions, isolate- and concentration-dependent differences in killing of the biofilms by tobramycin were apparent. However, tobramycin at concentrations up to 128 mg/l was unable to eradicate biofilms of any of the isolates tested, whether biofilms were grown aerobically or anaerobically. These results show that oxygen limitation may reduce, in a strain-dependent manner, the susceptibility to tobramycin of P. aeruginosa grown planktonically and as biofilms.