PURPOSE. To demonstrate the antimicrobial properties of riboflavin/ UVA (365 nm) against common pathogens. METHODS. One group of bacteria ( Pseudomonas aeruginosa [PA], Staphylococcus aureus [SA], and Staphylococcus epidermidis [SE]) was tested by using Kirby-Bauer discs with ( 1) empty disc (Control-C); ( 2) riboflavin 0.1% (B2); ( 3) riboflavin 0.1% previously activated by UVA (B2'); (4) UVA alone (UVA); ( 5) group 2+ additional UVA exposure (UVA + B2); and (6) group 3 + additional UVA exposure (UVA + B2'). In addition, another group of microbes was tested with the same approach: methicillin-resistant S. aureus (MRSA), multidrug-resistant P. aeruginosa (MDRPA), drug-resistant Streptococcus pneumoniae ( DRSP), and Candida albicans ( CA). The mean growth inhibition zone (GIZ) in square millimeters was measured around the discs. The mean standard deviation (MSD) was calculated to be 3.65 when alpha= 0.01. A mean deviation (MD) > MSD indicates a significant difference. RESULTS. In the first group, the GIZ was significantly greater after UVA (MD = 14.30), UVA+B2 (MD = 39.61), and UVA+B2' (MD = 40.45) when compared with C, B2, and B2'. UVA alone was less effective than UVA+B2 (MD = 25.31) and UVA+B2' (MD = 26.15). The second group demonstrated increased GIZ in UVA (MD = 6.98), UVA+B2 (MD = 17.80), and UVA+B2' (MD = 21.15) when compared with C, B2, and B2'. UVA alone was less effective against the second group of bacteria than was UVA+B2 (MD = 10.82) and UVA+B2' (MD = 14.17). CA did not show any GIZ after treatment. CONCLUSIONS. Riboflavin/UVA was effective against SA, SE, PA, MRSA, MDRPA, and DRSP, but was ineffective on CA and has the potential for use in treatment of microbial keratitis in the future.