Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI–TOF MS) has been widely used for microbial identification, because of its speed and accuracy, since its introduction to clinical microbiology laboratories. In this study, we evaluated the performance of ASTA MicroIDSys, a newly developed MALDI–TOF, and compared it with the widely used Bruker Biotyper. Microbial identification with the Bruker Biotyper system was performed by using a direct smear method and the Bruker Biotyper database (reference library version 6.0.0.0). The isolates were also tested in parallel, using the ASTA MicroIDSys system with a direct smear method and the MicroIDSys database, CoreDB v1.26. A total of 914 clinical isolates were recovered from the clinical specimens. Identical results with confidence scores (≥2.0, for the Bruker Biotyper) and acceptable scores (≥140 for the ASTA MicroIDSys) were obtained for 840 (91.9%) isolates. The minor errors were defined as misidentification at the species level, and the rate was 1.1% (9/792) for Bruker Biotyper and 1.6% (13/792) for ASTA MicroIDSys. Major errors were defined as misidentification at the genus level, and the rate was 0.3% (2/792) for both Bruker Biotyper and ASTA MicroIDSys. ASTA MicroIDSys showed reliable performance for microbial identification, which was comparable to that of the Bruker Biotyper. Therefore, ASTA MicroIDSys can be applied for the identification of microorganisms in clinical microbiology laboratories.