Fungal keratitis remains challenging due to its high incidence and potential complications that can lead to blindness. Early detection and early treatment of fungal keratitis are critical for avoiding vision-threatening complications. Cryotherapy can be a non-invasive and safe alternative therapy modality for fungal keratitis. This study aims to determine the effect of cryotherapy on corneal wound healing in a rat model of Aspergillus flavus keratitis using immunohistochemistry examination of TNF-α and IL-6 expression. Aspergillus flavus has injected intrastromal into the cornea of 35 Rattus norvegicus rats to establish fungal keratitis and then divided into five groups: baseline group, control group, natamycin group, cryotherapy group, and combination therapy group. Therapy was given five days after the fungal injection. Four days after therapy, the animals were euthanized and the eyeball was enucleated for immunohistochemistry examination to determine TNF-α and IL-6 expression. The differences in TNF-α and IL-6 expression were statistically analyzed with a significant level of p <0.05. The result showed that the highest IRS score of TNF-α and IL-6 expression was obtained in the baseline group. There was no significant difference in the intensity of TNF-α expression between the five groups (p-value 0.166). Meanwhile, statistical analysis of IL-6 expression revealed a significant difference in the intensity of IL-6 expression between the five groups (p-value 0.027), indicating cryotherapy can be considered as adjuvant therapy in the treatment of fungal keratitis.