Confocal microscopy offers several advantages over other conventional microscopic techniques as a tool for studying the interaction of bacteria with food and the role of food microstructure in product quality and safety. When using confocal microscopy, samples can be observed without extensive preparation processes, which allows for the evaluation of food without introducing artifacts. In addition, observations can be made in three dimensions without physically sectioning the specimen. The confocal microscope can be used to follow changes over a period of time, such as the development of the food structure or changes in microbial population during a process. Microbial attachment to and detachment from food and food contact surfaces with complex three-dimensional (3-D) structures can be observed in situ. The fate of microbial populations in food system depends on processing, distribution, and storage conditions as well as decontamination procedures that are applied to inactivate and remove them. The ability to determine the physiological status of microorganisms without disrupting their physical relationship with a food system can be useful for determining the means by which microorganisms survive decontamination treatments. Conventional culturing techniques can detect viable cells; however, these techniques lack the ability to locate viable cells in respect to the microscopic structures of food. Various microscopic methods take advantage of physiological changes in bacterial cells that are associated with the viability to assess the physiologic status of individual cells while retaining the ability to locate the cell within a food tissue system. This paper reviews the application of confocal microscopy in food research and direct observation of viable bacteria with emphasis on their use in food microbiology.