Infectious keratitis is a significant ocular disease that, if left untreated, can lead to blindness. Fungi are among the causative agents that can result in severe symptoms. Keratitis infections are prevalent globally, with a higher incidence reported in tropical and subtropical regions. The current research focused on the molecular diagnosis of fungal keratitis and its prevalence over a 3-year period in northeastern Iran. The study involved the collection of 38 corneal scraping specimens from the Eye Specialized Hospital of Khatam in Mashhad, northeastern Iran. These specimens were cultured on Sabouraud dextrose agar, and the isolates were identified using DNA-based techniques. Among the patients studied (n = 38), 22 (58%) cases were caused by Aspergillus species (A. flavus, n = 17, A. fumigatus, n = 3; A. terreus, n = 1; A. tubingensis, n = 1), seven (18%) by Neocosmospora species (N. falciformis, n = 4; N. solani, n = 3), three (7%) by Candida albicans, two (5%) by Fusarium annulatum, and one case each (2%) by Penicillium chrysogenum, Cladosporium cladosporioides, and Cytospora sp. In addition, one case had a combined infection of A. flavus and P. glabrum. The results indicate a higher incidence of fungal keratitis in males, particularly in the age range of 40-60 years. Aspergillus sp., and specifically A. flavus, had the highest prevalence. Cladosporium cladosporioides is reported for the first time in this area as causal agent of keratitis. Additionally, this is the first report of keratitis likely caused by Cytospora species.