Typing of bacterial subspecies is urgently needed for the diagnosis and efficient treatment during disease outbreaks. Physicochemical spectroscopy can provide a rapid analysis but its identification accuracy is still far from satisfactory. Herein, a novel feature-extractor-based fusion-assisted machine learning strategy has been developed for high accuracy and rapid strain differentiation using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and Raman spectroscopy. Based on this fusion approach, rapid and reliable identification and analysis can be performed within 24 hours. Validation on a panel of important pathogens comprising