Polluted waterbodies such as rivers provide a pathway or reservoir for bacterial resistance. We studied water quality and bacterial antibacterial resistance along the subtropical Qishan River in Taiwan as a case study of environmental resistance spread in a pristine to rural area. Human settlement densities increased generally from pristine mountain sites to the more polluted lowlands generally. Accordingly, as a working hypothesis, we expected antibacterial resistance level to increase towards downstream. We collected sediment samples from 8 stations along the Qishan river and where the Qishan river reaches the Kaoping river. The samples were processed in the lab for bacteriological and physicochemical analysis. Antibacterial resistance was tested with common antibacterials. A comparison was made among the sites where isolates began to occur at the upstream (site 1-6) with the downstream, including site 7 (Qishan town), site 8 (wastewater treatment plant) and site 9 (Kaoping river). The results of multivariate analysis for bacteriological and physicochemical parameters showed increasing water pollution levels downstream of the Qishan river. Bacterial isolates including Escherichia coli, Klebsiella pneumoniae, Serratia marcescens, Enterobacter sp., Acinetobacter sp., Staphylococcus spp. and Bacillus spp. were analyzed and tested in the study. Their percentage of occurrence varied at each site. The resistance level was determined from the growth inhibition zone diameter (disk diffusion) and the minimum inhibitory concentration (micro-dilution). The results indicated that antibacterial resistance was related to certain environmental factors. Besides, the usage pattern of different classes of antibacterials in different places could alter trends of their resistance. Bacteria were found with increased resistance to antibacterials used in agriculture through the downstream sites. The WWTP emitting wastewater was demonstrated to be a hotspot of resistance in aquatic environments. In conclusion, bacterial resistance against antibacterials from the Qishan river has become a potential public health threat. This study could assist authorities by providing a reference for water quality risk assessment and management in Kaohsiung city and southern Taiwan.