The aim of this study was to assess the antimicrobial resistance and the prevalence of genes determining the presence of extended-spectrum beta-lactamase (ESBL) enzymes in Escherichia coli isolated from two major rivers of the Podhale region in southern Poland. In total, 196 E. coli isolates were analyzed-98 from each river-Białka and Zakopianka, collected in 8 campaigns, over the period of two years. Antimicrobial resistance was assessed using disk diffusion method and PCR tests were conducted to detect the ESBL genes. In E. coli isolated from Białka, the resistance to amoxicillin/clavulanic acid was detected most frequently (54.08%) and ESBL was detected in 14.29% of strains. In strains isolated from Zakopianka, most frequent resistance was observed toward ticarcillin (51.02%), while ESBL was observed in 16.33% of isolates. In the total pool of isolates, the resistance to amoxicillin/clavulanic acid was most frequent (48.98% of isolates) and ESBL producers comprised 15.30% of E. coli isolates derived from both rivers. Multidrug resistance was observed less frequently in strains derived from Białka (4 isolates resistant to 10 and more antimicrobials) than from Zakopianka, where 10 isolates were resistant to 10 and more antibiotics. Out of the tested ESBL genes blaTEM was detected most frequently (45.4% of isolates), whereas blaCTX-M1 and blaCTX-M3 were recorded in one isolate.