Lake Zengena is one of the Crater Lake, which provides economic, social, and ecological benefits to the local community. The studies that were conducted in Lake Zangena concentrated mainly on Ecotourism. Information regarding the microbiology of the Lake Zengena is lacking. Thus, the aim of this study was determing the diversity and spatial variability of heterotrophic bacteria and evaluation of culture media for isolation of bacteria from lake zengena. Water samples were plated and incubated at 25oC for 48-72hrs on (TSA) and at 25oC for 5-7 days on R2A medium aerobically. Identification of was carried out according to Bergey’s manual of determinative bacteriology. Diversity indices were used to measure the diversity across site and between Media. Analysis of variance was used to test mean difference of Operational Taxonomic Units (OTUs) abundance between sites and between media type. A total of 200 isolates were picked from the two sites, purified, and grouped in to 76 OTUs based on morphological and biochemical characteristics. Eight OTUS were the most abundant and constitute about 22.5% of the total number of isolates and common to all sites and media types. Twenty-six OTUs (52 %) isolates were common to all media and 34 OTUs (57.7%) isolates were common to all sites. There were 27 unique OTUs in R2A and 24 unique OTUs in TSA medium. In case of sites, site 1 had 22 unique OTUs while site 2 had 20 unique OTUs. Variability in terms of media type was higher than spatial (sites). There was statistically significant difference in OTUs abundance between media type and sites, (p=0.001) R2A Site I (H=3.652) and R2A site II (H=3.569) exhibited higher diversity than TSA site I and TSA site II. Nine bacterial genera were identified, of which seven genera were present in all sites and media types were most dominant. Eight of identified genera were naturally occurring freshwater genera. The result of this study signifies conducting further research to identify each phenotype at species level to understand the ecological and economical role of the bacterial community. Key words: Bacteria, Diversity, Ecology, Isolates, Lake Zengena, spatial variability.