The challenges associated with anti-infective drug-discovery programmes can be tackled by combining several established and unprecedented hit-identification strategies with phenotypic antibacterial screening. I will illustrate this approach with a selection of un(der)explored targets. The first is a vitamin transporter from the energy-coupling factor (ECF) class, which is unique to Gram-positive bacteria. Here, we report on the structure-based virtual screening (SBVS), design, synthesis and structure–activity relationships of the first classes of selective, antibacterial inhibitors of the energycoupling factor (ECF) transporters with good in vitro and whole-cell activity and a good in vitro ADMET and in vivo PK profiles. A newly established cell-based uptake assay in Lactobacillus casei greatly facilitated our screening and hit-to-lead optimisation campaign. The second is the β-subunit of the bacterial DNA polymerase III (sliding clamp, DnaN), an attractive antibacterial target. We pursued several hit-identification strategies, including a SBVS campaign, affording novel chemotypes with micromolar affinity and promising antibacterial activity. Mode-ofaction studies confirmed DnaN as the molecular target. The new compound displays broad-spectrum antibacterial activity against mycobacteria, Gram-positive and Gram-negative pathogens also against multidrug-resistant bacteria with no cytotoxicity and good in vivo PK profiles. Finally, we succeeded in fragment merging and linking, affording highly selective and potent inhibitors of the extracellular metalloprotease and virulence factor of Pseudomonas aeruginosa, the elastase LasB. Multiparameter optimisation is currently ongoing based on extensive in vitro and ex vivo profiling, including the establishment of complex biological assays. Our approach promises to deliver the urgently needed anti-infective agents featuring both new chemical scaffolds and unprecedented modes of action. Multiparameter optimisation is currently ongoing based on extensive in vitro, wholecell, ex vivo and in vivo profiling, including the establishment of complex biological assays. A particular emphasis will be placed on the lead optimisation of frontrunners for permeation and achieving good lung exposure.