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The rapid identification of pathogens of bloodstream infections (BSIs) and the detection of antibiotic resistance markers are critically important for optimizing antibiotic therapy and infection control. The purpose of this study was to evaluate two approaches based on MALDI-TOF MS technology for direct identification of Gram-negative bacteria and automatic detection of <i>Klebsiella pneumoniae carbapenemase</i> (KPC) producers using the Bruker MBT Subtyping IVD Module in a large routine laboratory over a three-year period. MALDI-TOF MS analysis was performed directly from blood culture (BC) bottles following bacterial pellet recovery by Rapid MBT Sepsityper^® Kit and on blood agar 4-h subcultures. Automated detection of <i>bla</i>_KPC-carrying pKpQIL-plasmid by Bruker MBT Subtyping Module was evaluated in BCs tested positive to <i>K. pneumoniae</i> or <i>E. coli</i>. The results were compared with those obtained with conventional reference methods. Among the 2858 (93.4%) monomicrobial BCs, the overall species identification rates of the Rapid Sepsityper and the short-term subculture protocols were 84.5% (<i>n</i> = 2416) and 90.8% (<i>n</i> = 2595), respectively (<i>p</i> < 0.01). Excellent specificity for KPC-producers identification were observed for both MALDI-TOF MS protocols. The pKpQIL plasmid-related peak was detected in overall 91 of the 120 (75.8%) KPC-producing isolates. Notably, 14 out of the 17 (82.3%) <i>K. pneumoniae</i> isolates carrying <i>bla</i>_KPC variants associated with ceftazidime/avibactam resistance and tested negative by the immunocromatography assay, were correctly identified as KPC-producers by MALDI-TOF MS. In conclusion, combination of both Rapid Sepsityper and short-term subculture protocols may represent an optimal solution to promptly identify more than 95% of Gram-negative bacteria causing BSIs. MALDI Biotyper^® platform enabled a reliable and robust automated detection of KPC producers in parallel with species identification. However, integration of molecular or immunocromatographic assays are recommended according to local epidemiology.