The aim of this study was to investigate the extended-spectrum beta-laktamase activity (ESBL) and antibiotic resistance profile of 92 Escherichia coli strains isolated from bovine mastitic milk samples. Milk samples were obtained from dairy farms in Ankara, Balikesir and comm. ESBL screening test, ESBL phenotypic confirmation test and in vitro susceptibility test for twelve antibiotics were performed with Kirby Bauer disc diffusion method in E. con strains. Among the E. coli strains, respectively the highest resistance rates were detected against to erythromycin (69.6%), ampicillin (39.1%), tetracycline (34.8%), nalidixic acid (25.0%), chloramphenicol (22.8%), trimethoprim-sulfamethoxazole and amoxicillin-clavulanic acid (21.7%). Of all strains, 25.0% were found susceptible to all antibiotics and %54.3 of E. coli were resistant to two or more antibiotics. This was the first study that ESBL activity of E. coli isolated from mastitis was investigated in our country. However among the E. coli isolates, ESBL activity was not detected. This study showed that high rates of multidrug resistance developed in E. coli strains caused mastitis.