This study quantitatively examined short-term effects of 0.02% Mitomycin C (MMC) treatment on the nanostructural changes in human scleral collagen fibrils. Histologic analysis and non-contact mode atomic force microscopy (AFM) were employed to assess the ultrastructural changes in the morphological characteristics of human sclera before and after 0.02% MMC application for 1 and 3 min. The scleral collagen fibrils treated with 0.02% MMC for 1 min showed no significant change in the morphology of collagen fibrils, and a significant change (p < 0.05) in the thickness of scleral tissues and collagen density, compared to the controls. 0.02% MMC application for 3 min led to a significant increase (p < 0.001) in the mean fibril diameter (185.43 +/- 22.64 nm vs. 140.72 +/- 18.06 nm), thickness (0.81 +/- 0.03 mm vs. 0.54 +/- 0.05 mm) and collagen density (1.16 times), compared to the controls This study examined the nanostructural changes in the scleral collagen fibrils before and after MMC application by AFM technique combined with conventional histological analysis (Hematoxylin-eosin and Masson's trichrome). This result indirectly suggests that long-term MMC application might increase the incidence of complications like a scleromalcia.