Food safety and quality depend on raw materials characteristics and on the chemical, physical and biotechnological processes adopted during food transformation. Since a huge number of microorganisms are involved in food production, foodstuffs should be considered as complex matrices where any microbial component have a precise role and evolves in response to physical and chemical composition of food. Moreover, knowing the dynamics of microbial community involved in a food supply chain it is useful to reduce food spoilage, enhance industrial processes and extend products shelf-life. In a more comprehensive vision, a precise understanding of the metabolic activity of microorganisms can be used to drive biotransformation steps towards the improvement of quality and nutritional value of food. High Throughput Sequencing technologies (HTS) are nowadays an emerging and widely adopted tool for microbial characterization of food matrices. Differently from traditional culture-dependent approaches, HTS allows the analysis of genomic regions of the whole biotic panel inhabiting and constituting food ecosystems. Our intent is to provide an up-to-date review of the principal fields of application of HTS in food studies. In particular, we devoted major attention to the analysis of food microbiota and to the applied implications deriving from its characterization in the principal food categories to improve biotransformation processes.