Escherichia coli is an environmental pathogen causing clinical mastitis, a very important disease in dairy farms due to significant economic losses. This microorganism can use different mechanisms to colonize bovine mammary gland such as invasion cells, immune system evasion and biofilm production. The aim of this study was to investigate the biofilm formation by E. coli, isolated from milk of cows 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 study inclusion criteria were: mastitis control programs with data storage, average milk yield >20 kg, minimum of 200 lactating cows and use of machine milking. A total of 2,613 clinical mastitis milk samples was evaluated, during the period of September 2017 to September 2018. After clinical diagnosis, milk samples were collected aseptically. The isolation and identification were performed according to National Mastitis Council. E. coli was identified in 115 (4.4%) milk samples. For biofilm assay, we used 96-well polystyrene microplates and the 115 isolates were incubated in brain heart infusion (BHI) +1% glucose at 37°C for 18h without shaking. Under those conditions, 51 (44.3%) were not producers. Among the 54 remaining, 54 (47%) were classified as weak producers, 9 (7.8%) as moderate and only one (0.9%) as a strong producer. According to the results, it seems that biofilm formation is not a common characteristic among these isolates and does not play a key role in the establishment of mastitis in cows.