Studies on dental units (DUs) are conducted either for the prevention or the reduction of the density of bacterial contamination in dental unit waterlines (DUWLs). However, the existence of fungi in the these systems requires more attention. During dental treatment, direct contact with water contaminated with fungi such as Candida, Aspergillus, or inhalation of aerosols from high-speed drill may cause various respiratory infections, such as asthma, allergies, and wounds on mucose membranes, especially on immunocompromised patients and dentists. The aims of this study are to investigate the number and colonization of fungi in DUWLs in the city of Istanbul, Turkey. Water samples were collected from air-water syringes, high-speed drills, and inlet waters from 41 DUs. The aerobic mesophilic fungi count in high-speed drills was higher than inlet waters and air-water syringes. Non-sporulating fungi were found in 7 DUs. The isolated fungi were identified as Penicillium waksmanii, Cladosporium spp., Penicillium spp., Candida famata, Cryptococcus laurentii, Candida guilliermondii, Penicillium verrucosum, Aspergillus pseudoglaucus, Penicillium decumbens, and Acremonium sp. Some of these fungal genera are known as opportunistic pathogens that led to respiratory diseases such as allergic rhinits. This study shows the importance of regular control of mycological contamination on water at DUs.