BACKGROUND: The purpose of this study was to evaluate the antimicrobial activity of two nitric oxide donors, ie, S-nitrosoglutathione (GSNO) and S-nitroso-N-acetylcysteine (SNAC), against clinical isolates from patients with infectious keratitis. METHODS: Reference broth microdilution assays were performed to determine the minimum inhibitory and bactericidal concentrations for GSNO and SNAC against four American Type Culture Collection strains and 52 clinical isolates from patients with infectious keratitis as follows: 14 (26.9%) Pseudomonas species; 13 (25.0%) coagulase-negative Staphylococci; 10 (19.2%) Staphylococcus aureus; nine (17.3%) Serratia marcescens; and six (11.5%) Enterobacter aerogenes. Sterility control and bacterial growth control were also performed. RESULTS: SNAC showed lower minimum inhibitory and bactericidal concentrations than GSNO for all clinical isolates from patients with infectious keratitis. For Gram-positive bacteria, mean minimum inhibitory and bactericidal concentrations were 2.1 ± 1.3 and 8.6 ± 3.8 mM for SNAC and 4.6 ± 3.2 and 21.5 ± 12.5 mM for GSNO (P < 0.01). For Gram-negative bacteria, mean minimum inhibitory and bactericidal concentrations were 3.3 ± 1.4 and 6.1 ± 3.4 mM for SNAC and 12.4 ± 5.4 and 26.5 ± 10.1 mM for GSNO (P < 0.01). The minimum bactericidal to inhibitory concentration ratio was ≤8 in 100% of all isolates tested for SNAC and in 94.2% tested for GSNO. CONCLUSIONS: SNAC and GSNO had effective inhibitory and bactericidal effects against bacterial isolates from keratitis. SNAC showed greater antimicrobial activity than GSNO against all bacteria. Gram-positive bacteria were more susceptible to the inhibitory and bactericidal effects of the S-nitrosothiols.