We evaluated the occurrence of antibiotic-resistant bacteria, antibiotic-resistant gene, and metal concentration in a tropical river of Sri Lanka as a pre-emptive effort to understand the seasonal impact on their prevalence. Resistance for norfloxacin, ciprofloxacin, levofloxacin, kanamycin monosulfate (KM), tetracycline (TC), and sulfamethoxazole (ST) was measured with Kirby–Bauer disc diffusion method. The prevalence of Escherichia coli ranged from 10 to 27 CFU (colony-forming unit) ml−1 in Kelani River in Sri Lanka, and most of the E. coli isolates were resistant to more than one antibiotic. However, the resistance for TC and ST was much higher than other antibiotics. We found that the resistance percentage for older antibiotics like TC and ST was higher than the newer antibiotics. We detected genes that confer resistance to TCs, sulfonamides, β-lactams, and fluoroquinolones. Seasonal variation in the resistance of fluoroquinolones was much higher than the non-fluoroquinolones, but the effect was antagonistic. Overall, the significant seasonal variations imply the importance of mixed source and environmental conditions for development and transmission of antibiotic resistance.