The D-Ala-D-Ala adding enzyme (MurF) from Streptococcus pneumoniae catalyzes the ATP-dependent formation of the UDP-MurNAc-pentapeptide, a critical component of the bacterial cell wall. MurF is a potential target for antibacterial design because it is unique to bacteria and performs an essential non-redundant function in the bacterial cell. The recent discovery and subsequent cocrystal structure determination of MurF in complex with a new class of inhibitors served as a catalyst to begin a medicinal chemistry program aimed at improving their potency. We report here a multidisciplinary approach to this effort that allowed for rapid generation of cocrystal structures, thereby providing the crystallographic information critical for driving the inhibitor optimization process. This effort resulted in the discovery of low-nanomolar inhibitors of this bacterial enzyme.