Antibiotic resistance is the ability of microorganisms to resist the effect of an antibiotic to which they were once sensitive. Infections caused by bacteria resistant to first-line antibiotics and, more recently, last-line treatments, and even combination drugs could be fatal in case of minor bacterial infections or injuries. Co-trimoxazole (COT), a combination of two antifolate compounds - sulfamethoxazole and trimethoprim, is effective against Gram-negative and Gram-positive bacteria. There are multiple reports of co-trimoxazole resistance among clinical as well as environmental isolates of bacteria. We have investigated prevalence of COT resistance among bacterial isolates from urban aquatic environment and patients with urinary tract infections (UTI). Of the total bacterial cultures screened for the resistance 70-80 percent isolates were found to be positive. The transmission of ARGs among different bacterial species is a global human threat. This study aims at determining the prevalence and diversity of COT resistant bacteria in clinical and wastewater. Furthermore, this works highlights the acquisition of genetic factors viz. dfr and sul genes conferring resistance to COT among UTI causing bacterial pathogens and wastewater bacterial isolates. The study will further provide in-sights into the antibiotic resistance spread in clinical and environmental samples and how these two systems interlink.