New classes of antibiotics are urgently needed to counter increasing levels of pathogen resistance. Peptide deformylase (PDF) was originally selected as a specific bacterial target, but a human homologue, the inhibition of which causes cell death, was recently discovered. We developed a dual-screening strategy for selecting highly effective compounds with low inhibition effect against human PDF. We selected a new scaffold in vitro that discriminated between human and bacterial PDFs. Analyses of structure-activity relationships identified potent antibiotics such as 2-(5-bromo-1H-indol-3-yl)-N-hydroxyacetamide (6b) with the same mode of action in vivo as previously identified PDF inhibitors but without the apoptotic effects of these inhibitors in human cells.