Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) is commonly used by clinical microbiology laboratories to identify pathogens, despite some limitations of the technique. The Enterobacter cloacae complex (ECC) taxonomy has recently been expanded, leading to uncertain identification of some species within the ECC when commercial MALDI-TOF MS is used. This technique is especially unsuited in the case of E. hormaechei, the main species responsible for infections and one of the most prone, within the ECC, to acquire antibiotic resistance. Hence, rapid and reliable identification at the species level could improve patient management. Here, we evaluated the performance of the Bruker Microflex MALDI-TOF MS instrument to identify ECC isolates using two databases and algorithms in comparison to the hsp60 gene sequencing reference method: the Bruker database included in the MALDI Biotyper software and an extensive online database coupled to an original Mass Spectrometric Identification (MSI) algorithm. Among a panel of 94 ECC isolates tested in triplicate, the online database coupled to MSI software allowed the highest rate of identification at the species level (92%) compared to the MALDI Biotyper database (25%), especially for the species E. hormaechei (97% versus 20%). We show that by creating a database of MALDI-TOF reference spectral profiles with a high number of representatives associated with the performant MSI software, we were able to substantially improve the identification of the E. cloacae complex members, with only 8% of isolates misidentified at the species level. This online database is available through a free online MSI application (https://msi.happy-dev.fr/).