The prevalence of antibiotic residues and antibiotic-resistant bacteria in natural environments due to human and livestock waste emissions, pesticide runoff, and pharmaceutical effluents poses a serious threat to public health. Therefore, in this study, we aimed to examine the characteristics, including antibiotic susceptibility, biofilm formation ability, and virulence genes, of levofloxacin (LVFX)-resistant Escherichia coli isolated from the Tone River. Liquid chromatography-tandem mass spectrometry analysis revealed that the upstream, midstream, and downstream river water samples contained 2.2, 19.5, and 15.7 ng/L LVFX, respectively. Despite the very low concentration of LVFX, LVFX-resistant E. coli were isolated from the midstream and downstream regions. Based on the antibiotic sensitivity patterns, the following representative strains were selected: TLR101 and TLR104 (from midstream) and TLR105 and TLR108 (from downstream). These four isolates were LVFX and multidrug-resistant, with TLR101 showing high resistance to ampicillin and the others showing high resistance to chloramphenicol and tetracycline. Biofilm formation assays revealed that TLR104 and TLR108 exhibit very strong biofilm-forming abilities. One-step multiplex polymerase chain reaction (PCR) assays were used to detect various virulence genes, including eaeA, bfpA, invE, ipaH, stx1, stx2, elt, sth, aggR, and astA. A 100-bp astA gene product was detected in TLR104, and DNA sequence analysis confirmed this amplified PCR product, identifying it as a diarrheagenic E. coli strain. Overall, this study revealed the presence of multidrug-resistant diarrheagenic E. coli in the Tone River, underscoring the need for effective public health measures to prevent the release of untreated sewage and industrial waste into the river.