We examined the combined effect of fosfomycin and ofloxacin against Pseudomonas aeruginosa in biofilms by using an in vitro experimental system with a modified Robbins device. Sessile cells in a mature or immature biofilm, developed on a silicon disk, were used, and an ATP bioluminescence assay was employed to assess antibacterial effects. A synergistic effect of fosfomycin and ofloxacin was clearly detected when concentrations at which each drug independently produced no detectable decrease in the bioactivity of sessile cells were used. Exposure of the cells in a mature biofilm to fosfomycin at concentrations of one-eighth of the MIC to 10 times the MIC (6.25 to 500 micrograms/ml) and ofloxacin at three or 10 times the MIC (18.75 or 62.5 micrograms/ml) resulted in reduction of the bioactivity to 1.5 to 4.5% after 72 h. Young sessile cells in an immature biofilm were more susceptible to this combination therapy. With a combination of fosfomycin at three times the MIC and ofloxacin at three times the MIC, complete eradication was confirmed by both ATP assay and scanning electron microscopy.