Water is essential for life and it is an inorganic constituent of living matter. Water pipeline systems are sighted as problematic in aquatic habitats in which multiple pathogens are occupied including fungi. They have rigid cell walls containing glucans and chitin. The bodies of fungi comprise filaments called hyphae. These hyphae are split into a mat of interwoven single cells made of mycelium. Fungi can pollute the drinking water system and are responsible for biofilm formation. Biofilms are complex polymers containing many times their dry weight in water. Moisture is essential for biofilm formation. The occurrence of biofilms affects the quality of drinking water. Hence, the present study is aimed at recovering the fungi from drinking water samples and their biofilm formation in the water pipeline system. Drinking water samples such as mineral water, tap water, and RO-purified water are collected from different places. Fungi such as Aspergillus, Penicillium and Mucor were recovered from these samples and most species belong to Aspergillus and Penicillium. Further, the biofilm formation of fungi from cast iron in the pipeline system was detected using fluorescence microscopy and fluorescent in situ hybridization analysis.