The better understanding of the functioning of microbial communities is a challenging and crucial issue in the
field of food microbiology, as it constitutes a prerequisite to the optimization of positive and technological
microbial population functioning, as well as for the better control of pathogen contamination of food. Heterogeneity
appears nowas an intrinsic andmulti-origin feature of microbial populations and is a major determinant of
their beneficial or detrimental functional properties. The understanding of the molecular and cellular
mechanisms behind the behavior of bacteria in microbial communities requires therefore observations at the
single-cell level in order to overcome “averaging” effects inherent to traditional global approaches. Recent
advances in the development of fluorescence-based approaches dedicated to single-cell analysis provide the
opportunity to study microbial communities with an unprecedented level of resolution and to obtain detailed
insights on the cell structure, metabolism activity, multicellular behavior and bacterial interactions in complex
communities. These methods are now increasingly applied in the field of food microbiology in different areas
ranging from research laboratories to industry. In this perspective, we reviewed the main fluorescence-based
tools used for single-cell approaches and their concrete applications with specific focus on food microbiology.