The study aimed to investigate the microbiological and physicochemical quality of poultry drinking water in the Karak governorate, Jordan. Twenty-one water samples were collected from various poultry production locations, including broiler farms, layer farms, and local chicken butcher shops (Nattafat) in Karak. The susceptibility of the isolated gram-negative bacteria to commonly used antibiotics for poultry treatment, such as Cefotaxime, Amoxicillin-Clavulanate, Ciprofloxacin, and Gentamicin, were examined. The results revealed significant differences in the mean pH values and turbidity of water samples based on the source of collection. The values were 7.1 and 81.6 for broiler farms, 7.7 and 14.5 for layer farms, and 7.7 and 8.9 for butcher shops, respectively. Microbiological tests indicated that 66.7% of the total samples were contaminated with gram-negative bacteria, with the highest incidence of bacterial contamination found in broiler farms (85.7%). The isolated bacteria included Escherichia coli, Serratia odorifera, Stenotrophomonas maltophilia, Klebsiella oxytoca, Raoultella ornithinolytica, Salmonella enterica ssp Arizona, Kluyvera spp, Citrobacter koseri, and Citrobacter amalonaticus. Evaluation of the isolated bacteria's susceptibility to selected antibiotics showed that all bacterial isolates exhibited resistance against Amoxicillin-Clavulanate, Ciprofloxacin, and Gentamicin, except for E. coli, which showed partial sensitivity to gentamicin. All isolated bacteria, except S. odorifera, were susceptible to the cefotaxime antibiotic. In conclusion, the study emphasizes the importance of implementing a quality control system in poultry management to improve the quality of drinking water, reduce bacterial infections, and mitigate the emergence of multidrug-resistant (MDR) bacteria.