Four slime-producing uropathogenic Escherichia coli strains were used to investigate the activity of fosfomycin and N-acetylcysteine (NAC) against biofilms developed on 96-well polystyrene tissue culture plates. Biofilms aged, respectively, 5 (initial) and 48 h (mature) and two fosfomycin concentrations (128 and 2000 mg/l) were used. The effect of various levels (0.007-8 mg/ml) of NAC alone and in combination with fosfomycin on the formation or disruption of biofilms was assessed. Following exposure to the drugs, the percentage of residual slime relative to the control, ranged from 62.5-100 to 26.2-64.1% in the presence of 0.007 and 8 mg/ml of NAC. After treatment of pre-formed biofilms with NAC at the highest concentrations used, the remaining exopolysaccharide matrix was reduced to 25-68% of the amount found with the untreated control. Exposure to fosfomycin at 2000 mg/l reduced biofilms 40-57 and 41-49% for the initial and mature forms, respectively. Fosfomycin was more active at 2000 mg/l combined with NAC 2 mg/ml. Under these conditions initial and mature biofilms were reduced 66-80 and 60-73%, respectively. NAC, when used in combination, enhanced fosfomycin bactericidal activity producing a 99-99.9% reduction in viable cells. Fosfomycin and NAC at concentrations achievable in urine displayed a synergistic effect promoting both the formation of biofilms and reduction of sessile cell viability.