Biosensors are devices which combine a biochemical recognition element with a physical transducer. There are various types of biosensors, including electrochemical, acoustical, and optical sensors. Biosensors are used for medical applications and for environmental testing. Although biosensors are not commonly used for food microbial analysis, they have great potential for the detection of microbial pathogens and their toxins in food. They enable fast or real-time detection, portability, and multipathogen detection for both field and laboratory analysis. Several applications have been developed for microbial analysis of food pathogens, including E. coli O157:H7, Staphylococcus aureus, Salmonella, and Listeria monocytogenes, as well as various microbial toxins such as staphylococcal enterotoxins and mycotoxins. Biosensors have several potential advantages over other methods of analysis, including sensitivity in the range of ng/mL for microbial toxins and <100 colony-forming units/mL for bacteria. Fast or real-time detection can provide almost immediate interactive information about the sample tested, enabling users to take corrective measures before consumption or further contamination can occur. Miniaturization of biosensors enables biosensor integration into various food production equipment and machinery. Potential uses of biosensors for food microbiology include online process microbial monitoring to provide real-time information in food production and analysis of microbial pathogens and their toxins in finished food. Biosensors can also be integrated into Hazard Analysis and Critical Control Point programs, enabling critical microbial analysis of the entire food manufacturing process. In this review, the main biosensor approaches, technologies, instrumentation, and applications for food microbial analysis are described.