Bacterial DNA replication requires an orchestration of protein-protein and protein-DNA interactions to faithfully replicate DNA prior to cell division. One protein essential to this process, the β2-clamp, interacts with several proteins via a conserved binding site and mechanism. We have been probing this binding site as a target for novel antibiotics across Gram-negative bacteria. Six classes of novel compounds selected via a DNA-encoded chemical screening program were found to target the E. coli β2-clamp. We have characterised the interaction to the protein-binding pocket of the β2-clamp using X-ray crystallography and biophysical assays. The compounds, as first-generation hits, have affinities ranging from mid μM to mid nM against various Gram-negative β2clamps, inhibit in vitro DNA replication at low μM concentrations and showed promising antimicrobial activity. These inhibitors were ineffective against the human homologue, PCNA, and some were non-toxic to human cells. We are currently investigating the mechanism of action of these inhibitors using single-molecule in vivo imaging techniques. These are promising inhibitors of the β2-clamp and provide excellent scaffolds for optimization into antimicrobial compounds.