Background and Aims: The incidence of fungal infections in Saudi Arabia is increasing with an increase in the population of high-risk patients. Still, studies highlighting the impact of environmental fungal agents on infections in Saudi are limited, and none have examined the Medina region despite the city’s stature, diversity, and travel flow. This study aims to determine the fungal distribution across the Medina region. Methods: A randomized 212 indoor samples were collected based on analyzed data from 1374 questionnaires. The prevalence of participants to fungal infection was 19.2% and 26.3% in the risk group. Fungal species were identified via macroscopic and microscopic examination. Results: Two hundred and three (96%) samples showed fungal growth. Yeast identified were 78 (34%) Candida species, 56 (25%) non-Candida species. The study identified 13 genera and 20 species for mould, including 38 (17%) Aspergillus spp., 20 (9%) Rhizopus spp. 9(4%) holoarthic/enteroatric conidiogenesis, 7 (3%) Mucor spp. 4 (2%) of both Penicillium spp. and Neurspora spp. and only 2(1%) of Madurella spp., 3(1%) Epidermophyton spp., 2 (1%) Lichtheimia spp., and 1(1%) Peacilomyces spp. Conclusions: This study identified Aspergillus spp. as the most frequent fungi across the Medina region. The characterization of indoor fungal environment enables healthcare practitioners to anticipate fungal infection for individuals at risk. These findings can aid the prediction and prevention of fungal infections and fungal sensitization among the high-risk populations.