Abstract Background To harmonize with the EUCAST breakpoints, the French Society of Microbiology introduced a change in the inhibition diameter breakpoint (17 mm versus 20 mm previously) of temocillin. We assessed the impact of the new breakpoints on categorizing susceptibility of Enterobacterales to temocillin. Methods This was a multicentric retrospective study including all Enterobacterales isolates routinely tested for temocillin susceptibility with the disc diffusion method between 1 January 2016 and 31 July 2022 in four centres. Categorization using the breakpoints of 20 mm (French guidelines CA-SFM/EUCAST 2020 v.1.1) and 17 mm (French guidelines CA-SFM/EUCAST 2021 v1.0 and EUCAST guidelines v11.0) was performed. Results Overall, 36 416 Enterobacterales isolates were included. The overall rate of temocillin resistance decreased from 11.3% to 4.7% (relative difference of 58.5%) when using the 17 mm breakpoint instead of the 20 mm breakpoint, respectively. The relative change ranged from −44.0% in Klebsiella aerogenes to −72.7% in Klebsiella oxytoca. The median inhibition diameter was 23 mm (IQR 21–25). The isolates with a diameter of 20 mm appeared overrepresented, whereas those with a diameter of 18 and 19 mm were underrepresented. We therefore reviewed the diameters between 18 and 21 mm of 273 isolates. Thirty-two (11.7%) of them categorized as susceptible at first measure were controlled resistant at second measure. Conclusions The new breakpoint induced a decrease in the rate of isolates categorized as resistant to temocillin, increasing therapeutic choice including for Extended-spectrum beta-lactamase-producing Enterobacterales (ESBL-PE). We suggest the bias in measuring the inhibition diameter is probably related to the fact that temocillin is considered remarkably stable against broad-spectrum β-lactamases.