The demand for rapid, consistent and easy-to-use techniques for detecting and identifying pathogens in various areas, such as clinical diagnosis, the pharmaceutical industry, environmental science and food inspection, is very important. In this study, the reference strains of six food-borne pathogens, namely, Escherichia coli 0157: H7 ATCC 43890, Cronobacter sakazakii ATCC 29004, Salmonella Typhimurium ATCC 43971, Staphylococcus aureus KCCM 40050, Bacillus subtilis ATCC 14579, and Listeria monocytogenes ATCC 19115, were chosen for scanning electron microscopy (SEM) and energy dispersive X-ray (EDX) analysis. In our study, the time-consuming sample preparation step for the microbial analysis under SEM was avoided, which makes this detection process notably rapid. Samples were loaded onto a 0.01-µm-thick silver (Ag) foil surface to avoid any charging effect. Two different excitation voltages, 10 kV and 5 kV, were used to determine the elemental information. Information obtained from SEM-EDX can distinguish individual single cells and detect viable and nonviable microorganisms. This work demonstrates that the combination of morphological and elemental information obtained from SEM-EDX analysis with the help of principal component analysis (PCA) enables the rapid identification of single microbial cells without following time-consuming microbiological cultivation methods.