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Abstract Background Fungal keratitis is one of the major causes of visual impairment worldwide. However, the effectiveness of corneal collagen cross-linking (CXL) for fungal keratitis remains controversial. In this study, we developed an in vitro and an in vivo models to assess the efficacy of CXL for Fusarium keratitis. Methods The effect of in vitro CXL fungicidal was evaluated on the cultures of Fusarium solani which were exposed to irradiation for different durations. Viability of fungal was appraised under four conditions: no treatment (control); CXL: UVA (365 nm)/riboflavin; riboflavin and UVA (365 nm). Each batch of sterile plate culture was irradiated for different CXL durations. The in vivo Therapeutic effect was studied on a mouse keratitis model. The animals were divided randomly into three groups: group A with no treatment (control); Group B with CXL treatment for two minutes and group C with CXL treatment for three minutes. The CXL procedure was performed 24 h post inoculation in each group. All mice with corneal involvement were scored daily for 7 days and 10 days after infection. Corneals were extracted at various time points for quantitative fungal recovery. Histological evaluations were conducted to calculate the number of polymorphonuclear cells. Results Viability of fungal decreased significantly in CXL group with 30-min irradiation compared with that in control, riboflavin and UVA groups (P