Rapid identification of bacterial pathogens facilitates earlier optimization of antibiotic treatment and reduces morbidity and mortality in sepsis patients. The aim of this research was to design an in-house chemical-free method for direct bacterial identification in positive blood culture (BC) broths and to compare the performance of this method with that of the commercial Sepsityper® kit. The overall species identification rates for the in-house and Sepsityper methods were 88.4% and 85.8%, respectively (n = 190). Among 146 facultative anaerobes, 92.5% and 95.9% were identified to the species level using the in-house and Sepsityper methods, respectively. For 32 anaerobic bacteria, the in-house method showed a higher species identification rate (75.0%) than the Sepsityper method (53.1%). The in-house method correctly identified more Bacteroides species (100.0%) than the Sepsityper method (18.2%). Our novel in-house method and the Sepsityper method showed a high accuracy for direct bacterial identification in positive BC broths using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.Copyright © 2020. Published by Elsevier B.V.