Ultraviolet light C (light wavelength 200 nm to 290 nm) has been shown to kill cultures of antibiotic resistant strains of bacteria such as methicillin-resistant Staphylococcus aureus. To evaluate the ability of ultraviolet light C to reduce the amount and type of bacteria present in chronically infected ulcers, as well as to establish the test-retest reliability of the semi-quantitative swab technique, a prospective, one-group, pre-post treatment study was conducted among patients receiving treatment in several in- and outpatient facilities and nursing homes. Individuals with chronic ulcers exhibiting at least two signs of infection and critically colonized with bacteria (n = 22) received a single 180-second treatment using an ultraviolet light C lamp (wavelength = 254 nm) placed 1 inch from the wound bed. Semi-quantitative swabs taken immediately before and after UVC treatment were used to assess changes in the bacterial bioburden present within the wound bed. Results demonstrated excellent test-retest reliability of the semi-quantitative swab technique used to evaluate the type and amount of bacteria present in chronic wounds (Cohen's kappa = 0.92). Assessment of wound bioburden using semi-quantitative swabs revealed a statistically significant (P <0.0001) reduction in the relative amount of bacteria following a single treatment of ultraviolet light C. The greatest reduction in semi-quantitative swab scores following ultraviolet light C treatment were observed for wounds colonized with the bacteria Pseudomonas aeruginosa and wounds colonized with only one species of bacteria. Significant (P <0.05) reductions in the relative amount of bacteria also were observed in 12 ulcers in which methicillin-resistant Staphylococcus aureus was present. These results confirm previous laboratory studies and demonstrate that ultraviolet light C can kill bacteria such as Pseudomonas aeruginosa, Staphylococcus aureus, and methicillin-resistant Staphylococcus aureus present in superficial layers of chronic wounds.