Seventy Fusarium isolates derived from human keratomycosis were identified based on partial sequences of the β-tubulin (β-TUB) and translation elongation factor 1α (EF-1α) genes. Most of the isolates were confirmed as members of the F. solani species complex (75.71%), followed by the F. dimerum species complex (8.57%), the F. fujikuroi species complex (8.57%), the F. oxysporum species complex (4.29%) and the F. incarnatum-equiseti species complex (2.86%). A combined phylogenetic tree was estimated including all the 70 isolates. Isolates belonging to different species complexes formed separate clades. In this study, we also report the first isolation of F. napiforme from human keratomycosis. A new method based on a specific EcoRI restriction site in the EF-1α gene was developed for the rapid identification of F. solani. In vitro antifungal susceptibilities of the isolates to seven antifungals were determined by broth microdilution method. Terbinafine, natamycin and amphotericin B proved to be the most effective drugs, followed by voriconazole. The minimal inhibitory concentrations of clotrimazole, econazole and itraconazole were generally high (≥64 μg ml−1). The interactions between the two most effective antifungals (natamycin and terbinafine) were determined by checkerboard microdilution method. Synergism (71.8%) or no interaction (28.2%) was revealed between the two compounds.