Albicidin is a potent antibiotic and phytotoxin produced by Xanthomonas albilineans which targets the plant and bacterial DNA gyrase. We now report on a new albicidin derivative which is carbamoylated at the N-terminal coumaric acid by the action of the ATP-dependent O-carbamoyltransferase Alb15, present in the albicidin (alb) gene cluster. Carbamoyl-albicidin was characterized by tandem mass spectrometry from cultures of a Xanthomonas overproducer strain and the gene function confirmed by gene inactivation of alb15 in X. albilineans. Expression of alb15 in Escherichia coli and in vitro reconstitution of the carbamoyltransferase activity confirmed albicidin as the substrate. The chemical synthesis of carbamoyl-albicidin finally enabled us to assess its bioactivity by means of in vitro gyrase inhibition and antibacterial assays. Compared to albicidin, carbamoyl-albicidin showed a significantly higher inhibitory efficiency against bacterial gyrase (∼8 vs 49 nM), which identifies the carbamoyl group as an important structural feature of albicidin maturation.