This study aims to isolate and characterize efficient biohydrogen generating facultative anaerobic bacteria from various samples, viz., biogas plant (BGP), municipal sewage (MS), and dairy industry treatment plant (DTP). The physicochemical properties of various untreated anaerobic sludge samples reflect the anoxic state and appropriateness of the substrate for separating biohydrogen generating bacteria. The biohydrogen producing bacterial strains were separated from methanogens using the heat-treatment method. The facultative anaerobic bacterial load of heat-treated test samples was determined viz., 27.2±0.57×10⁶ (BGP), 21.8±0.43×10⁶ (MS), and 18.6±0.92×10⁶ (DTP) CFU mL^-1 (Colony forming unit), which decreased from the total anaerobic bacterial load of untreated anaerobic sludge viz., 32.1±0.28×10⁶ (BGP), 42.2±0.16×10⁶ (MS), and 34.7±0.12×10⁶ (DTP) CFU mL^-1. The 28 predominant bacterial isolates strains were isolated from the heat-treated test samples. All 28 bacterial strains were identified using microscopic and biochemical techniques. Biohydrogen producing potential bacterial strains were screened using the Hungate technique with glucose as a carbon source. Among them, 12 strains were capable of producing biohydrogen, among these 5 strains being excellent biohydrogen producers. Based on the16s rRNA molecular sequencing, the 5 selected biohydrogen generating organisms were authenticated as viz., Salmonella bongori (MZ636759), Escherichia coli (MZ636716), Staphylococcus hominis (MZ636713), Yersinia enterocolitica (OM009292), and Shewanella oneidensis (MZ636800). The gas composition study by GC-TCD in a fermentative medium shows that Shewanella oneidensis (MZ636800) could produce the best biohydrogen (111.4±8.3 mLH₂/L), followed by Salmonella bongori (MZ636759) with 98.1±2.9 mL H₂/L and Escherichia coli (MZ636716) with 86.7±6.2 mLH₂/L.