MraY (phospho-N-acetylmuramoyl-pentapeptide-transferase) inhibitory natural products are attractive molecules as candidates for a new class of antibacterial agents to combat antimicrobial-resistant bacteria. This paper develops a strategy for comprehensive *in situ* evaluation of a build-up library to streamline analogue preparation and directly assess biological activities. Applying this to MraY inhibitors, 686 compounds were identified, with promising analogues showing potent, broad-spectrum antibacterial activity *in vitro* and *in vivo*. MraY-analogue complex structures reveal unique binding modes. The strategy's generality is demonstrated using tubulin-binding natural products.
