BackgroundWe investigated the presence of heteroresistance against both tigecycline and colistin inAcinetobacter baumanniiand then evaluated the effectiveness of combined antibiotic treatment given the existence of discrete tigecycline- and colistin-resistant subpopulations.MethodsWe performed population analysis profiling (PAP) to evaluate the degree of composite heteroresistance inA. baumanniiisolates, with the extent of this resistance quantified using subsequent antibiotic susceptibility testing. We then evaluated the amino acid sequence of PmrBAC and the relative mRNA expression levels ofpmrB.Finally, we investigated the combined antibiotic efficacy of tigecycline and colistin in multiple-heteroresistant isolates using dual PAP and in vitro time-killing assays.ResultsAll tigecycline-heteroresistantA. baumanniiisolates, with the exception of one colistin-resistant isolate, were also heteroresistant to colistin. Evaluations of the colistin-resistant subpopulations revealed amino acid alterations in PmrA and PmrB and increased expression ofpmrB. All tigecycline-resistant subpopulations were susceptible to colistin, and all colistin-resistant subpopulations were susceptible to tigecycline. Dual PAP analysis using tigecycline and colistin showed no heteroresistance, and in vitro time-killing assays revealed that a combination of these two antibiotics effectively eliminated the bacterial cells.ConclusionOur results suggest that multiple heteroresistance to tigecycline and colistin is highly prevalent amongA. baumanniiclinical isolates and that these resistant subpopulations exist independently in single multiple heteroresistant isolates. Therefore, our findings may explain the success of combined antibiotic therapies in these infections.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12929-023-00914-6.