The Song River in Dehradun, Uttarakhand, India, travels from Dhanaulti to Narendra Nagar and originates as a spring-fed stream on the southern slopes of the Himalayan range's Radi Top of Missouri ridge. Fungi are the principal decomposers and work with bacteria in the environment, thus actively participating in the circulation of environmental nutrient cycles such as nitrogen and phosphorus. The aim of this research is the Isolation and screening of Aqua-borne fungi from different locations of Song River. Twenty water samples were gathered from several locations of song rivers in a 1-liter sterile bottle and used 500 mg, Chloramphenicol to inhibit the growth of bacteria, and 1ml and 0.1 ml water samples were poured into PDA, SDA, Rose Bengal agar Medium and the poured plate incubated 4-10 days for fungi growth. After that, the pure culturing method and microscopic observation with Lacto cotton blue strain were used. We reported some species of fungi Rhodotorula sp. 45%, A. Candidus 70%, Rhizopus sp. 35%, A. Niger 85%, and A. flavus 75%, are found in the Song River sites. One of the most recent discoveries on fungus that demands special attention is the frequency of emerging and opportunistic infections in raw water sources. Because fungus can impact water quality in a number of ways, the microbiota of water should be considered when assessing the microbiological safety and quality of drinking water.