Fluorescence in situ hybridization (FISH) offers considerable potential for visualizing microorganisms in food matrices. It enables the non-destructive counting, localization, and distribution analysis of beneficial, spoilage, and pathogenic microbes. Recent advances in technology and methodological refinements have further improved the applicability of FISH in the investigation of food-associated microorganisms. Gaining deeper insights into the functions of microbial communities is a critical and complex challenge in food microbiology. This understanding is essential for optimizing the activity of beneficial and technologically useful microbes and for improving the control of foodborne pathogens. FISH is particularly valuable for detecting bacteria in samples where culturable microbes constitute only a small fraction of the total population. It has been successfully used for the targeted detection of spoilage organisms, the early identification of contamination sources, and the monitoring of food pathogens and parasites in production processes. This chapter provides an up-to-date overview of FISH protocols used in the microbiological analysis of food.