The in vitro activity of fosfomycin (an antibiotic that has clinically not been widely used in Nigeria) against 516 clinical bacteria isolates and the screening for the presence of R plasmid conferring resistance to fosfomycin among the test bacteria isolates were determined. In the presence of added glucose-6-phosphate (25 μg/ml) to the growth medium, all the isolates were inhibited at fosfomycin minimum inhibitory concentrations (MIC) of ≤ 32–64 μg/ml. Without the glucose-6-phosphate, fosfomycin had MIC75, MIC70, MIC48, and MIC20 at 64 μg/ml against Escherichia coli, Pseudomonas aeruginosa, Klebsiella spp., and Serratia spp., respectively, while the rest of the strains maintained about the same susceptibility as in the presence of glucose-6-phosphate. An R plasmid of about 59 megadaltons in size, conferring resistance to streptomycin, ampicillin, carbenicillin, and fosfomycin, was obtained from a Serratia liquifacens isolated in an area where fosfomycin had not been clinically used.