<p>Antimicrobial resistance (AMR) represents a critical public health challenge, with surface waters serving as reservoirs for antibiotic-resistant bacteria (ARB). Among these, gram-negative enteric bacteria (GNEB) are recognized as major carriers of resistance genes and frequent causes of human infections. As a major tourism destination in Thailand, Phuket Province is likely to face increasing AMR-related issues. This study investigates water quality and the prevalence of ARB in the Bang Yai Canal, a key urban waterway in Phuket, and its transition into Phuket Bay. Water samples were collected from nine stations during the dry and rainy seasons of 2024. Total heterotrophic bacterial counts and GNEB resistant to amoxicillin, tetracycline, norfloxacin, and meropenem were examined in relation to water quality parameters. Results revealed significant spatial variation, with urban areas contributing substantially to ARB prevalence. Amoxicillin-resistant bacteria were the most prevalent, particularly among GNEB, while meropenem-resistant bacteria were consistently detected at most stations despite their low abundance. Seasonal variations indicated higher bacterial abundance upstream during the dry season and downstream during the rainy season, potentially driven by tourism and runoff dynamics. The coastal station exhibited a notably high proportion of antibiotic-resistant marine heterotrophic bacteria. Redundancy analysis identified turbidity and dissolved oxygen as significant factors influencing bacterial counts. Cluster analysis grouped stations based on water quality, with upstream and coastal sites exhibiting distinct profiles. This study underscores the critical role of urban activities in ARB dissemination and highlights the environmental and public health implications of ARB in coastal ecosystems, necessitating targeted mitigation and monitoring strategies.</p>