The expression of enzymes of the OXA-48 carbapenemase group is difficult to detect by phenotypic methods owing to frequent low levels of carbapenem resistance and negative results with some screening methods. Temocillin has been shown to be a good option for phenotypic screening as it is hydrolysed by the OXA-48-group enzymes, whereas ESBLs, AmpC and some other carbapenemases have a lower hydrolytic effect on this antimicrobial. However, no epidemiological cut-off for temocillin is available. To evaluate temocillin MICs in relation to the presence or absence of genes encoding ESBLs and carbapenemases in Escherichia coli and Salmonella enterica. In this study, 111 E. coli and 102 S. enterica isolates, including WT and well-characterized ESBL-, AmpC- or carbapenemase-producing isolates, were tested by three independent laboratories. MICs were determined according to the CLSI guidelines by agar dilution with the test range from 0.5 to 512 mg/L temocillin and WGS was performed and analysed with ResFinder. Some overlap was detected between temocillin MICs for WT and ESBL- or AmpC-producing isolates. However, isolates carrying genes encoding carbapenemases showed a broader range of MICs for both E. coli and S. enterica. Higher MICs were observed for the OXA-48 group, VIM and some NDM-producing isolates, whereas isolates harbouring KPC enzymes showed low MICs. The results indicate that temocillin MICs enable phenotypic distinction between strains producing OXA-48-group enzymes and both WT susceptible and ESBL/AmpC-carrying isolates, whereas the distinction from other carbapenemases likely requires genotypic testing.