Objective Extraintestinal Escherichia coli isolates are the most common gram-negative pathogens in humans and cause urinary tract infections, sepsis, neonatal meningitis, and others. The aim of this study was to investigate the rates of antibiotic resistance and virulence factors (kpsM II, neuc K1, hlyF, fyuA, afa/draBC, sat, chuA, fimH, tsh, yfcv, ibeA, traT, iucD, usp, iutA, cnf1, hlyA, papC, sfa/focDE, and ompT) of E. coli strains isolated from blood and urine samples. Methods A total of 150 E. coli strains isolated from blood and urine samples sent to the Microbiology Laboratory, Faculty of Medicine Hospital, Selcuk University were included in the study. The identification and antibiotic susceptibility tests were performed with the VITEK 2 automated system. Multiplex polymerase chain reaction was used to detect the virulence genes. Results Although the highest antibiotic resistance rate found was against ampicillin (73.3%), the lowest rates were against ertapenem and meropenem (0.7%). Extended-spectrum β-lactamase positivity was 38% in E. coli blood isolates and 29% in urine. The highest rates of virulence genes were detected in fimH gene (92%). iutA gene was 91.3%, traT 76%, fyuA 50%, chuA 54.7%, iucD 46.7%, ompT 32.7%, yfcv 31.3%, hlyF 28.7%, sat 22%, papC and sfa/focDE 20%, kpsM II 19.3%, neuc K1 14.7%, tsh 13.3%, cnf1 6.7%, afa/draBC 6%, ibeA 5.3%, usp 4.7%, and hlyA 3.3%. kpsM II, tsh, hlyA, papC, sfa/focDE, and ompT genes were higher in blood isolates. Conclusion High antibiotic resistance rates and virulence genes were detected in E. coli strains in Konya, Turkey. This is the first study in Turkey where both a large number and a variety of virulence factors were investigated and compared. Multicenter studies are needed to better understand E. coli virulence.