The identification of microorganisms directly from blood cultures using MALDI-TOF MS has been shown to be the most impacting application of this methodology. In this study, a novel commercial method was evaluated in four clinical microbiology laboratories. Positive blood culture samples (n = 801) were processed using a rapid BACpro^® II kit and then compared with the routine gold standard. A subset of monomicrobial BCs (n = 560) were analyzed in parallel with a Sepsityper^® Kit (Bruker Daltonics, Bremen, Germany) and compared with the rapid BACpro^® II kit. In addition, this kit was also compared with two different in-house methods. Overall, 80.0% of the monomicrobial isolates (609/761; 95% CI 71.5–88.5) were correctly identified by the rapid BACpro^® II kit at the species level (92.3% of the Gram negative and 72.4% of the Gram positive bacteria). The comparison with the Sepsityper^® Kit showed that the rapid BACpro^® II kit generated higher rates of correct species-level identification for all categories (p > 0.0001), except for yeasts identified with score values > 1.7. It also proved superior to the ammonium chloride method (p > 0.0001), but the differential centrifugation method allowed for higher rates of correct identification for Gram negative bacteria (p > 0.1). The percentage of accurate species-level identification of Gram positive bacteria was particularly noteworthy in comparison with other commercial and in-house methods.