ABSTRACT Members of the Enterobacter cloacae complex (ECC) are almost ubiquitous in nature and can act as pathogens. In this study, we used a polyphasic taxonomy approach to establish the accurate taxonomic position of six strains within the ECC. Notably, the 16S rRNA gene-based phylogeny failed to group all Enterobacter species into a monophyletic cluster. As an alternative to this, we explored genome sequence-based phylogenetic approaches. The bac120 gene-based phylogeny successfully grouped all Enterobacter species into a monophyletic cluster, although some species-level clustering conflicted with average nucleotide identity (ANI) values. Furthermore, the Enterobacter -specific core gene phylogeny resolved all species, aligning with ANI results. Three strains were identified as Enterobacter asburiae , while strain P99 was classified as “ Enterobacter xiangfangensis ” and strain C45 as Enterobacter quasihormaechei . Conversely, strain A-8 T formed a distinct cluster in all phylogenies, with ANI and digital DNA-DNA hybridization (dDDH) values below the species threshold (<92% and <44%, respectively) with all known Enterobacter species. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry biotyper results confirmed that all strains belonged to the ECC. However, the comparison of mean spectrum profile of strain A-8 T with those of other strains revealed the presence of 18 unique peaks, highlighting its distinct protein profile. Based on comprehensive genotypic and phenotypic characterizations, we propose that strain A-8 T is a new species of the genus Enterobacter , which is named Enterobacter pasteurii sp. nov. The type strain is A-8 T (CIP 103550 T ; ATCC 23355 T ; DSM 26481 T ; and WDCM 00082 T ). This study advances our understanding of the ECC, emphasizing the need for multidimensional taxonomic techniques and contributing to the better management of microbial resource centers. IMPORTANCE Accurate taxonomy is essential for microbial biological resource centers, since the microbial resources are often used to support new discoveries and subsequent research. Here, we used genome sequence data, alongside matrix-assisted laser desorption/ionization time-of-flight mass spectrometer biotyper-based protein profiling, to accurately identify six Enterobacter cloacae complex strains. This approach effectively identified distinct species within the E. cloacae complex, including Enterobacter asburiae , “ Enterobacter xiangfangensis ,” and Enterobacter quasihormaechei . Moreover, the study revealed the existence of a novel species within the Enterobacter genus, for which we proposed the name Enterobacter pasteurii sp. nov. In summary, this study demonstrates the significance of adopting a genome sequence-driven taxonomy approach for the precise identification of bacterial strains in a biological resource center and expands our understanding of the E. cloacae complex.