Background . Antimicrobial resistance (AMR) in ESKAPE pathogens, notably in carbapenem-resistant Acinetobacter baumannii, is a pressing global health concern, contributing to significant AMR-associated mortality. Efflux pump overexpression plays a crucial role in the multidrug resistance (MDR) of A. baumannii , but the identification of efflux-overexpressing strains remains challenging. Methods . We evaluated the performance of an efflux pump inhibition assay in comparison with RT-qPCR, to assess efflux pump overexpression in a collection of clinical A. baumannii strains. Sixty MDR strains and a wild one were analyzed. Results . The inhibition assay was positive for 34 and negative for 27 strains, respectively. Results were in agreement with RT-qPCR in 67.2% of the tested strains (39/58). Cohen's kappa showed a fair agreement between inhibition assay and RT-qPCR (Cohen’s kappa = 0.37). Inhibition by NMP, but not PAβN, was in moderate agreement with RT-qPCR (Cohen’s kappa = 0.41).Furthermore, gpi alleles were associated with efflux pump overexpression ( p = 0.03), suggesting potential links between genetic diversity and efflux pump overexpression. RT-qPCR showed a significant efflux pump overexpression for 4 (16.7%) out of 24 inhibition assay negative strains. Conclusions . Our findings support the ability to reliably rule out efflux pump overexpression in A. baumannii strains displaying a negative result with an inhibition test relying on NMP. However, confirmatory analyses are necessary for strains that test positive.