Microbial pathogens cause a quarter of all deaths worldwide annually due to deadly infectious diseases. Nevertheless, the fast and precise identification of pathogens remains one of the most challenging tasks in the medical sector. Early identification and characterization of microbes through medical diagnosis could pave the way for specific treatment strategies that could dramatically improve infection management, reduce healthcare costs, mitigate increasing antimicrobial resistance, and save numerous lives. To date, numerous traditional and molecular methods have been employed to diagnose illnesses with proven accuracy, reliability, and efficiency. Here, we have reviewed the most reliable tools that are prerequisites for the rapid detection of microbes. In particular, the remarkable roles of surface-enhanced Raman scattering, Fourier-transform infrared, electrochemical impedance, near-infrared, and MALDI-TOF/TOF in the identification and characterization of pathogenic microbes are discussed in detail. The approaches described herein cover broad ranges of biomedical applications, including the diagnosis of clinical infectious diseases, epidemiology, detection of vector-borne diseases, food security, phytosanitary monitoring, biosensing, and food- and waterborne pathogen detection. Considering the current pandemic outbreak, this review briefly emphasizes the importance of rapid detection and upgraded tools for early diagnosis to prevent the loss of lives.