Purpose: Antifungal efficacy of photochemical cross-linking (PACK-CXL) with 0.1% and 0.25% riboflavin was evaluated with a comparative in vitro study.Methods: Candida albicans and Aspergillus fumigatus ATCC reference strains, Candida parapsilosis, Aspergillus fumigatus, Fusarium solani, Scedosporium apiospermum, and Alternaria alternata strains isolated from keratitis cases were chosen as targeted microorganisms. Unique black plate method was developed in polystyrene microplates. Riboflavin suspensions in 0.1% and 0.25% were separately added into inoculated wells. Non-inoculated wells were filled by black colored dye in order to protect treated wells from reflection of UV treatment. After ultraviolet A (UVA) treatment, each well was evaluated by microbiological culture in order to count viable fungal colonies. Fungal killing rate was calculated by comparing fungal counts (CFU/mL) before and after UVA application of riboflavin-added wells.Results: Four different fungal inoculum concentrations of targeted microorganisms, including 10(4), 10(3), 10(2), and 10(1) CFU/mL, were assayed. PACK-CXL with 0.25% riboflavin was found to be highly effective on fungal cells even in 10(4) CFU/mL of concentration.Conclusions: PACK-CXL appears as a promising treatment option for difficult-to-treat cases of fungal keratitis and 0.25% riboflavin concentration increases fungicidal effect of the procedure dramatically.