There are conflicting reports on the antibacterial activity of ascorbate; all at concentrations much higher than the typical in human plasma, but that can be reached in urine. The effect of 10 mM ascorbate (in itself not inhibitory) along with antibiotics, was tested both in Mueller-Hinton broth (MHb) and in synthetic human urine (SHU), against resistant isolates ofEscherichia colifrom lower urinary infections. The activity of nitrofurantoin and sulfamethoxazole was higher in SHU than in MHb; minimal inhibitory concentrations (MICs) in SHU with ascorbate were below typical urinary concentrations. For other antibiotics, MICs were the same in MHb vs. SHU, with no effect of ascorbate in MHb; but in SHU with ascorbate, MICs of ciprofloxacin and gentamicin also went below reported urinary concentrations, with a lesser effect with norfloxacin and trimethoprim, and none with ampicillin. The effect of ascorbate was independent of oxygen and not related to the susceptibility of each strain to oxidative stress. Ascorbate oxidizes during incubation in SHU, and bacterial growth partially prevented oxidation. These results suggest that 10 mM ascorbate can enhance the inhibitory activity of antibiotics upon resistant strains in urine. Clinical experimentation with ascorbate–antibiotic combinations against urinary infections caused by resistant bacteria is warranted.