The Karasu River (Sinop, Turkey) was sampled for bacteriological analyses and investigation of antibiotic resistance at the bacterial isolates. In the study, the physical properties and numbers of total aerobic (TA), total coliform (TC), faecal coliform (FC), faecal Streptococci (FS) and Escherichia coli in water samples were investigated. E. coli strains were subjected to antibiotic susceptibility testing, antibiotic resistant genes, virulence genes and plasmid DNA analysis and fingerprinting techniques. The bacteriological analysis results showed that the difference between TA, TC, FC and FS numbers (p > 0.05) was not significant for a total of four stations. The Multiple Antibiotic Resistance Index (MARI) values of tested strains were found in range from 0.4 to 0.7. The antibiotic resistance genes of strains; tetA (33.9%), blacym‐2 (12.5%) and cat1 (16.7%) were screened by PCR method. Out of a total of 57 E. coli isolates, 11 E. coli strains were positive for the eae (19.6%) gene, representing atypical EPEC, whilst only one strain was positive for the eae and stx1 (1.7%) gene, representing STEC. Present study has shown that the aquatic environments can be reservoirs of pathogenic E. coli strains. The study also indicated that PCR‐based techniques might especially be helpful in the rapid identification of multidrug‐resistance analysis and virulence genes of strains. Karasu stream, which is important in Sinop province, Turkey, was investigated for the first time in this study in terms of a molecular microbiological perspective.