Monitoring drinking water quality is essential for protecting public health, particularly in regions dependent on surface water reservoirs. Morbe Dam is the primary source of potable water for Navi Mumbai and surrounding areas. Conventional culture-based methods may not reliably distinguish closely related environmental bacteria, highlighting the need for molecular confirmation during microbial surveillance. Water samples were collected from multiple locations within Morbe Dam and analysed using conventional microbiological methods, including selective culture, Gram staining, and biochemical characterization. Presumptive Salmonella isolates were further identified by 16S rRNA gene amplification, sequencing, and BLAST analysis. Antimicrobial susceptibility testing was performed using the Kirby–Bauer disk diffusion method, followed by MIC determination for selected antibiotics. Conventional culture and biochemical tests yielded isolates presumptively identified as Salmonella spp. However, 16S rRNA gene sequencing did not confirm Salmonella. Instead, BLAST analysis of the partial 16S rRNA gene sequence revealed approximately 96% sequence similarity to Ralstonia mannitolilytica, indicating that the isolate belonged to the genus Ralstonia and was most closely related to R. mannitolilytica. The sequence was deposited in GenBank under Accession No. MW647908. Antimicrobial susceptibility testing demonstrated resistance to multiple β-lactam antibiotics. No inhibition was observed for amoxicillin/clavulanic acid and cefazolin at concentrations up to 256 µg/mL, while MICs for ceftazidime and cefuroxime were 16 µg/mL and 8 µg/mL, respectively. This study demonstrates that reliance on phenotypic methods alone may lead to misidentification of environmental bacterial isolates. Molecular analysis demonstrated that the isolate belonged to the genus Ralstonia and was most closely related to Ralstonia mannitolilytica, rather than Salmonella. These findings support the integration of molecular diagnostics into routine environmental water surveillance and emphasize the importance of monitoring antimicrobial-resistant opportunistic pathogens in potable water sources.