The emergence of antibiotic-resistant bacteria is posing a challenge and threat to the environment especially their prevalence in water bodies. One of the most prevalent bacteria in E.coli is a gut bacteria found in the gastrointestinal tract of animals. This is a pilot study carried out to identify the presence of such antibiotic-resistant E. coli in surface waters of a prominent river in Trivandrum city known as Killi river. The isolated bacteria were confirmed to be E. coli by biochemical and molecular analysis. We also wanted to look for the effect of methanolic extract of the paradise tree, Simarouba gluca on such multidrug-resistant bacteria. Our studies have analyzed the phytochemical composition of methanolic extract of S. gluca and confirmed it by TLC analysis. We also looked for the antibacterial effect of the extract using the agar diffusion method and show that the methanolic extract of S. gluca exhibits good antimicrobial activity against antibiotic-resistant E. coli. This study is the first of its kind to have demonstrated the effectiveness of the plant extract as a potent antimicrobial agent against environmental strains of E.coli isolated from Killi river that are resistant to some of the common antibiotics used for the treatment of a wide variety of infections in both humans and animals. This study would offer a better option for designing plant-based compounds while preparing drugs for combating these kinds of bacteria.