In healthcare settings, hospital water and water-related devices can act as a reservoir for waterborne infections. Potable water, sinks, faucet aerators, showers, tub immersion, toilets, dialysis water, water baths, eyewash stations, and dental-unit water stations have all been linked to nosocomial outbreaks. This study aimed to determine the microbial profile and pattern of antibiotic resistance in the water supply of a tertiary care hospital in Uttarakhand. This is a 1-year prospective study which was carried out by the Department of Microbiology and Immunology at Sri Mahant Indersh Hospital (SMIH), Dehradun. A total of 154 water samples were collected from the AC outlets, ventilators in the Intensive care unit (ICUs), Operation theatre (OTs), and High dependency unit (HDUs), scrub stations, pantry, and blood bank, patient's bathroom, private ward, septic ward, labour room, transplant unit, laboratory, scope rinse water, the dialysis unit and tank throughout the hospital, including tap water (pre and post flush [25%]), tap swabs (24%), drinking water (9%), AC outlets (13%), and other areas (3%). 30 of the 154 (19.5%) water samples tested were culture-positive. The most contaminated water samples were tap swabs (27%, n = 8/30). A total of nine organisms were isolated, of which the most predominant was Pseudomonas aeruginosa (40%; 12/30), followed by Legionella pneumophila (13%; 4/30), Acinetobacter baumanii (10%; 3/30), Klebsiella pneumoniae (10%; 3/30), Escherichia coli (7%; 2/30), Enterococcus faecalis (7%; 2/30), Aspergillus flavus (7%; 2/30), Stenotrophomonas (3%; 1/30), and Fusarium spp. (3%; 1/30). Gram-negative bacilli and non-lactose fermenting (GNB and NLF) showed a high rate of contamination, 53.3% (n = 16/30). P. aeruginosa showed resistance to gentamicin and amikacin (42%), imipenem (50%), levofloxacin (58%), and colistin (25%), while Acinetobacter baumanii showed resistance to gentamicin and amikacin (67%), minocycline (63%), and levofloxacin, imipenem and colistin (33%). The study's findings show that a variety of microorganisms are contaminating hospital water supplies and can be a source of hospital-acquired infections. A suitable and robust surveillance program for hospital water supplies, as well as strict adherence to infection control practices, is strongly advised.