We aimed to investigate the occurrence of acquired AmpC β-lactamases (qAmpC), and characterize qAmpC-producing Enterobacteriaceae from different non-clinical environments in Portugal. We analysed 880 Enterobacteriaceae resistant to third-generation cephalosporins recovered from 632 non-clinical samples [healthy human and healthy animal (swine, chickens) faeces; uncooked chicken carcasses; aquatic and trout aquaculture samples]. Bacterial and qAmpC identification, antibiotic susceptibility, clonal (PFGE, MLST) and plasmid (S1-/I-CeuI-PFGE, replicon typing, hybridization) analysis were performed using standard methods. The occurrence of qAmpC among Enterobacteriaceae from non-clinical origins was low (0.6%; n = 4/628 samples), corresponding to CMY-2-producing Escherichia coli from three healthy humans (HH) and one uncooked chicken carcass (UCC). We highlight a slight increase in CMY-2 human faecal carriage in the two periods sampled [1.0% in 2013-2014 versus 0% in 2001-2004], which is in accordance with the trend observed in other European countries. CMY-2-producing E. coli belonged to B22-ST4953 (n = 2, HH), A0-ST665 (n = 1, HH) or A1-ST48 (n = 1, UCC) clones. blaCMY-2 was identified in non-typeable and IncA/C2 plasmids. This study is one of the few providing an integrated evaluation of the qAmpC-producing Enterobacteriaceae occurrence, which was low, from a very large collection of different non-clinical origins. Further surveillance in contemporary collections can provide an integrated epidemiological information of potential shifts in reservoirs, transmission routes and mechanisms of dissemination of blaqAmpC in non-clinical settings.