MurA catalyzes the first step of peptidoglycan (PG) biosynthesis and is a validated target for the development of new antimicrobial agents. In this study, a library of 49 plant flavonoids and their synthetic derivatives were evaluated for their inhibitory properties against MurA fromEscherichia coli. The compounds were tested with and without preincubation and with the addition of DTT to understand the mechanism of inhibition. Thirteen compounds were identified as reversible, time-dependent inhibitors, with inhibition levels ranging from 480 nM to 57 μM, and ampelopsin as the most potent compound. To investigate the major pharmacophore elements responsible for the activity, 2D-QSAR and docking analyzes were performed. The results showed that the catechol moiety and an additional aromatic system were the most important features contributing to the activity of the compounds. However, most of the compounds did not show antibacterial activity againstE. coli andStaphylococcus aureusstrains, suggesting that their inhibitory activity against MurA may not be strong enough to induce antibacterial effects. Nevertheless, our results suggest that flavonoids are a promising starting point to develop new inhibitors of MurA and show great potential for further steps in drug development.