Microbiological contamination of food remains a critical global public health concern, contributing to millions of foodborne illness cases each year. Traditional diagnostic methods, particularly culture-based techniques, have been widely employed but are often limited by low sensitivity, insufficient specificity, and lengthy turnaround times. Recent advances in molecular biology, biosensor technology, and analytical chemistry have enabled the development of more rapid and precise diagnostic tools. Among these, Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) has emerged as a transformative method for microbial identification. This review provides a comprehensive overview of the current applications of MALDI-TOF MS in the diagnosis of microbiological contamination in food. The method offers rapid, accurate, and cost-effective identification of microorganisms and is increasingly used in food safety laboratories for the detection of foodborne pathogens, ensuring the safety and quality of food products. We highlight the fundamental principles of MALDI-TOF MS, discuss its methodologies, and examine its advantages, limitations, and future prospects in food microbiology and quality assurance.