The objective of this study was to evaluate the impact of urbanization and seasonal changes on the prevalence of antibiotic-resistant bacteria in different aqueous environments. To this end, bacteria were isolated from three different water sources: the River Hooghly in Kolkata, River Kangsabati and groundwater from Kharagpur, West Bengal over three seasons: post-monsoon, winter and summer in 2012-2013. A total of 163 Gram-negative bacteria were isolated from the River Hooghly (n = 138), River Kangsabati (n = 13) and groundwater (n = 12). Antibiotic susceptibility testing was done using 12 antibiotic discs. The percentages of multiple antibiotic-resistant (MAR) bacteria at the three sampling locations were found to be 71.01 % (98/138) for River Hooghly, 15.38 % (2/13) for River Kangsabati and 8.33 % (1/12) for groundwater. Prevalence of MAR bacteria with respect to the three seasons were the following: 73.58 % in post-monsoon, 59.26 % in winter and 53.57 % in summer. Antibiotic resistance index (ARI) was calculated for each location and each season. In general, ARI values for all the River Hooghly samples were >0.2 while those for the River Kangsabati and groundwater in Kharagpur were always <0.2 indicating greater exposure to antibiotics and subsequent resistance in bacteria from the River Hooghly compared to the other two locations. In addition, percentage of MAR and ARI values followed the trend: post-monsoon > winter > summer. This may be due to the additional terrestrial resistants that get swept along with surface runoff during the monsoons.