Enterobacter cloacae complex has been increasingly recognized as a nosocomial pathogen representing the third major Enterobacteriaceae species involved with infections. This study aims to evaluate virulence and antimicrobial susceptibility of subpopulations generated from macrocolonies of NDM-1 producing Enterobacter hormaechei clinical isolates. Biofilm was quantified using crystal violet method and fimbrial genes were investigated by PCR. Susceptibility of antimicrobials, alone and combined, was determined by minimum inhibitory concentration and checkerboard assays, respectively. Virulence and efficacy of antimicrobials were evaluated in Galleria mellonella larvae. Importantly, we verified that some subpopulations that originate from the same macrocolony present different biofilm production ability and distinct susceptibility to meropenem due to the loss of blaNDM-1 encoding plasmid. A more in-depth study was performed with the 798 macrocolony subpopulations. Type 3 fimbriae were straightly related with biofilm production; however, virulence in larvae was not statistically different among subpopulations. Triple combination with meropenem–rifampicin–polymyxin B showed in vitro synergistic effect against all subpopulations; while in vivo this treatment showed different efficacy rates for 798-1S and 798-4S subpopulations. The ability of multidrug resistant E. hormaechei isolates in generating bacterial subpopulations presenting different susceptible and virulence mechanisms are worrisome and may explain why these infections are hardly overcome.