Background Dermatophytes are group of fungi that cause superficial infections via enzymes that degrade keratin in human skin. Several factors, including climate, gender, age, lifestyle, human migration, cultural habits, and socioeconomic status influence the prevalence of dermatophyte infections. We analyzed the prevalence of dermatophyte isolates in a hospital in Eastern Saudi Arabia from 2000 to 2019. Methods The data on fungal cultures were obtained from the Laboratory Information System of the Mycology Laboratories at Johns Hopkins Aramco Healthcare, and were used for the analysis. Fungal isolates were examined microscopically for the presence of specialized hyphal structures and conidia. The Vitek® MS microbial identification system (biomerieux) was used if the culture type was not identified microscopically. Results Among the 10,021 samples analyzed, 3040 (30.33%) were positive for fungi and only 398 (3.97%) were dermatophytes. Microsporum species was the most common dermatophyte accounting for 50.5% (n = 201) followed by trichophyton with 36.9% (n = 147). The most common positive samples were scrapping (251, 63%) and hair (68, 17%). Culture positivity relative to the age groups revealed a cluster of positive dermatophyte species in children < 10 years of age with 215 (54%) of all cases and among 10–19 years of age with 60 (15) of the cases (p < 0.001). Microsporum species were the prevalent dermatophytes in patients  < 10 years of age, while Epidermophyton species were the most frequent dermatophyte species in age groups 10–19, 20–29, and 30–39 years. However, Trichophyton species were the most frequent dermatophyte species in individuals 70–79 years. The percentage of Microsporum and Trichophyton species decreased significantly over time (p < 0.001). In addition, there was a significant seasonal variation in relation to Trichophyton species. A comparison between the most frequent species showed that there was no difference in relation to gender, but there was a difference in relation to the specimen type and age group. Conclusion Dermatophytosis was common among children and adolescent with the most common samples were scrapping and hair. There was a significant reduction in Microsporum and Trichophyton species over time.

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