Identifying the mechanism of action of antimicrobials remains a challenge and is a major limitation for the drug discovery process. Here, Pogliano and colleagues test the ability of bacterial cytological profiling (BCP) to determine the mechanism of action of antimicrobials. BCP involves the treatment of bacterial cells with a compound and the subsequent analysis of morphological changes to identify the cellular pathway targeted. Using this technique, the authors could distinguish between inhibitory compounds that target different cellular pathways (for example, protein synthesis or DNA replication), as they generated distinct cytological profiles. Moreover, they used BCP to show that the antibacterial compound spirohexenolide A probably kills bacterial cells by disrupting the cytoplasmic membrane. Thus, BCP could be used to rapidly characterize the mechanism of action of antimicrobials and, in combination with other approaches, to determine the precise target in the pathway.