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Extended-spectrum β-lactamase (ESBL)-producing Gram negative bacteria are becoming increasingly important in veterinary and human medicine because they can hydrolyze the third generation β-lactams, penicillins, and monobactams. The aim of this study was to identify ESBL-producing Enterobacteriaceae in raw cow milk samples from northeast Brazil. Twenty-six bacterial isolates belonging to the Enterobacteriaceae family were obtained from milk samples from 257 cows with subclinical mastitis. Using microbiological tests, 53.85% (14/26) were identified as Escherichia coli, 15.38% (4/26) as Proteus mirabilis, 26.92% (7/26) as Klebsiella spp., and 3.85% (1/26) as Citrobacter spp. Of all the isolates, 61.54% (16/26) were positive in the ESBL screening test, of which 12.5% (2/16) were positive in the double-disc synergy test using three types of cephalosporins and amoxicillin/clavulanic acid. The two isolates were identified as Klebsiella spp. Among all the isolates, 53.85% (14/26) were positive for one or both ESBL-encoding genes, blaSHV and blaTEM; among these, 71.43% (10/14) were identified as E. coli. This study demonstrates that ESBL-producing bacteria can be found in raw cow milk from northeast Brazil. Cows with subclinical mastitis should be recognized as reservoirs of these strains, which can propagate to humans., (© 2023. The Author(s) under exclusive licence to Sociedade Brasileira de Microbiologia.)