Karoon River is the most important river in Iran that supplies the drinking and industrial water to many cities and villages in Khuzestan. As a consequence of hospital and municipal wastewater discharge into this river, antibiotic resistance will be increased in microbial inhabitants of this ecosystem. Furthermore, microbial load is also undergone continual variation. The aim of this study was to evaluate seasonal microbial quality and antibiotic resistance in bacterial isolates. For this purpose, five stations were selected and samples were harvested during four seasons. Bacterial count was performed through culturing on Mueller-Hinton agar, and bacterial isolates were identified. Antibiotic resistance profiles of isolates as well as resistance to Hg were investigated. As a result, it was found that the least quality was in summer season while the best quality was in winter that is related to the reduction of water volume and recreational activities in summer and increasing rain and dilution of contaminants in winter season. All isolates were sensitive to Hg while antibiotic resistance and multidrug resistance were found in bacterial isolates. Based on the obtained results, it can be concluded that the microbial quality of Karoon River has variations depending on the season and it is necessary that be monitored in order to control and prevent epidemic bacterial infections and antibiotic resistance distribution.