In microbiology, fluorescence tools have undoubtedly proven their performance to analyze in real-time, non-invasively and quantitatively, the structure, composition, processes, and dynamics of microbial communities. Importantly, the available fluorescence-based methods enable to decipher multiscale events implicated in the failures and successes of antibiotics treatments. The goal of this article is to review the large range of fluorescence techniques and probes and their interest (i) to study bacterial cells viability in vitro and in vivo under antibiotics exposure, (ii) to elucidate the mechanisms of action of these drugs, and (iii) to dissect bacterial mechanisms to resist and tolerate antibiotics action.