Background: Fosfomycin is an old broad-spectrum bactericidal agent. It has been suggested that fosfomycin can be introduced in the treatment of urinary tract infections resulted from multi-drug resistant organisms. Objective: In this study, the effect of fosfomycin on uropathogenic Escherichia coli and its ability to produce biofilm was studied. Methodology: Sixty uropathogenic E. coli isolates were collected. Antibiotic susceptibility testing was performed using disk diffusion method. The minimum inhibitory concentration (MIC) of fosfomycin was measured using agar dilution technique. Biofilm formation was studied using tissue culture plate method. The effect of sub-inhibitory concentrations of fosfomycin on the biofilm forming ability of the isolates was assessed. Results: High rates of resistance were reported for most of the tested antibiotics except for fosfomycin, to which all isolates were susceptible, and imipenem, to which 58 (96.67%) isolates were susceptible. MIC testing revealed 16.67% resistance rate to fosfomycin. Twelve (20%) isolates were found to be biofilm forming. Ten of them (83.33%) lost their ability to produce biofilm under the effect of fosfomycin. Conclusions: This study revealed a high antibacterial efficacy of fosfomycin against uropathogenic E. coli ability to produce biofilm in vitro. Further research studies are required to assess its ability to abolish biofilm formation on urinary catheters.