The ability to generate antimicrobial reactive-oxygen species (ROS) by reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is critical for the host to defend against invading microbes. We have demonstrated the application of confocal autofluorescence microscopy and Raman microspectroscopy to characterize dynamically the phagocytic process of living macrophages in a label-free manner. In particular, we visualized the translocation of NADPH oxidases in livin macrophages that are undergoing phagocytosis of invading yeasts and monitored dynamically the change at the molecular level of single intraphagosomal yeasts caused by phagocytic ROS.