This study aims to evaluate the positive predictive value (PPV) and the negative predictive value (NPV) of Vitek2 in detecting extended-spectrum beta-lactamase (ESBL) phenotypes when compared to a manual confirmatory test as gold standard. A sample of Escherichia coli, Klebsiella spp and Proteus mirabilis isolates were collected by 5 laboratories in the Emilia-Romagna Region (Italy). Vitek2 appears to be an accurate tool to detect ESBL phenotypes of E. coli isolates; some concern remains about its performance with the other bacterial species, especially P. mirabilis.