This research aimed to discover the macroscopic, microscopic, and physiological characteristics and the genus of heterotrophic bacteria found in the schmutzdecke or biofilm layer in slow sand filters. The isolation of heterotrophic bacteria in the schmutzdecke applied the pour plate and quadrant streak method, while the characterization used macroscopic, microscopic, and physiological tests. Samples were taken from the schmutzdecke on top of the filter layer, and they were diluted 3, 4, and 5 times, then grown in Nutrient Agar media in order to isolate heterotrophic bacteria. The results of the research were analyzed using the identification manual books titled Bergey’s Manual of Determinative Bacteriology, 9th edition and Manual for The Identification of Medical Bacteria, 3rd edition. These manuals show the names of the genus of bacteria in the schmutzdecke layer. On the basis of the identification results from macroscopic, microscopic, and physiological tests, there were 4 dominant genera out of 18 living isolates obtained from the schmutzdecke layer, namely Kurthia gibsonii, Bacillus badius, Bacillus firmus, and Bacillus lentus. The similarity percentage of these 4 isolates was 83%, 81%, 85%, and 77% respectively.