Sediment is a special habitat in freshwater ecosystem and harbors a variety of heterotrophic bacterial communities. Studying bacterial diversity has an important role for a better understanding of the functioning of aquatic systems however, studies on biodiversity have mainly focused on macroorganisms and little attention have been directed toward microorganisms. The objective of this study was to investigate the diversity and spatial variability of heterotrophic bacteria and evaluate efficacy of two culture media on isolation of bacteria from the sediment of Lake Zengena. Sediment samples were collected from two sampling sites of Lake Zengena via corer sampling device aseptically. Sediment samples were plated in triplicate aerobically on TSA media for three days and R2A media for five days at 25 o C following a serious of serial dilution technique. A total of 200 isolates were picked, purified and grouped into 73 Operational Taxonomic Units (OTUs) according to Bergey’s manual of determinative bacteriology from the basis of the result of gram stain, colony morphology and biochemical tests. Diversity indices were used to measure the diversity between sites and media type. Analysis of variance was used to test the mean difference of Operational Taxonomic Units (OTUs) abundance between sites and media. Seventeen of the 73 OTUs were common for both sites and had 61(30%) isolates and fourteen OTUs were common for both media types and represented by 53 (26.5%) isolates. There were 29 and 27 unique OTUs in site one and two respectively. Moreover, R2A media had 29 unique OTUs while TSA media had 22 unique OTUs and variation in both sites and growth media indicate different bacterial abundance. Members of the cultured bacterial community in this study were identified into 9 genera belonged to 4 phyla: Proteobacteria, Actinobacteria, Firmicutes and Bacteroidetes. There was a statistically significant difference in OTUs abundance between sites at (p=0.014) and media type at (p=0.02). OTUs diversity was higher in R2A media site one (H=3.269) than TSA media site one (H=3.082). Furthermore, OTUs diversity was higher in R2A media site two (H=3.167) than TSA media site two (H=3.054) due to varied composition of organic compounds in the growth media. The result of this study revealed that sediment bacterial communities in Zengena Lake were diverse and mainly composed of phyla that are typical to freshwater sediment. Future work needs to identify each OTUs at the species level to understand the ecological role of the bacterial community.