With the rapid development of multi-resistant bacteria, the arsenal of effective antibiotics to treat infections is dramatically shrinking. This crisis is further intensified by safety concerns with a class of front-line antibiotics, the fluoroquinolones (FQs), which received several black box warnings by the US food and drug administration (FDA) due to severe side effects. A restricted use of FQs is in particular a drawback for the treatment of Gram-negative strains which are only addressed by a limited number of alternative antibiotics capable of entering their almost impermeable cell membranes. Thus, to keep these desperately needed antibiotics within the first-line of defense against pathogenic bacteria, it is crucial to decipher the unwanted off-targets in human cells, to understand the origin of side effects and develop chemical strategies to enhance selectivity towards bacterial cells.