Bovine mastitis is one of the major diseases associated with the dairy industry, causing great economic losses. Escherichia coli is the most prevalent microorganism in clinical mastitis, causing severe symptoms and even death. In Brazil, little is known about the genetic background of E. coli isolates causing mastitis, and if these isolates can cause illness in the human hos, such as a zoonotic agent. In this study, we investigated the occurrence of several virulence-factor encoding genes in E. coli isolates from milk of cows diagnosed with clinical mastitis. This study was approved by the UNESP’s Animal Use Ethics Committee (N 0136/2017). We used 10 different dairy herds, selected by convenience, and inclusion criteria were: mastitis control programs with data storage, average milk yield >20 kg, minimum of 200 lactating cows and use of machine milking. After clinical diagnosis, milk samples were collected aseptically. The isolation and identification were performed according to National Mastitis Council. The occurrence of genes frequently used for diarrheagenic E. coli (DEC) pathotypes identification (escN, bfpA, stx1, stx2, elt, est, ipaH, aatA, aggR, aaiA and daaE) and involved in extra-intestinal E. coli (ExPEC) pathogenicity (sfaDE, fimH, iha, ecpA, papA, papC, hlyA, cnf1, sat, vat, iroN, irp2, iucD, ireA, sitA, ibe10, traT, ompT, afaBC, and cdt) were searched by PCR in the 93 E. coli isolates obtained in this study. One atypical EPEC (escN⁺/bfpA^-) and one atypical EAEC (aaiA⁺/aggR^-) were detected. Regarding the genes associated with ExPEC pathogenicity, we observe the presence of genes encoding for: adhesins (fimH = 98%; ecpA = 48.4%), siderophores (irp2 = 8.6%; sitA = 8.6%), toxins (vat = 3.2%; cdt = 2.2%; hlyA = 5.4%, invasin (ibe = 2.2%), and protactins (ompT = 19.4%; traT = 59.1%). We concluded that the majority of the isolates cannot be classified in the distinct DEC pathotypes, but possess pathogenic potential to cause extra intestinal disease.