Aminoacyl-tRNA synthetases (aaRSs) are promising drug targets due to their essential roles in protein translation. Although current inhibitors primarily occupy one or two of the three substrate binding sites on aaRSs, we report here the structure-based design of the first class of triple-site aaRS inhibitors by targeting <i>Salmonella enterica</i> threonyl-tRNA synthetase (<i>Se</i>ThrRS). Competition of our compounds with all three substrates on <i>Se</i>ThrRS binding was confirmed <i>via</i> isothermal titration calorimetry assays. Cocrystal structures of three compounds bound to <i>Se</i>ThrRS unambiguously confirmed their substrate-mimicking triple-site binding mode. Compound <b>36j</b> exhibited the best enzyme activity against <i>Se</i>ThrRS with IC<sub>50</sub> = 19 nM and <i>K</i><sub>d</sub> = 35.4 nM. Compounds <b>36b</b>, <b>36k</b>, and <b>36l</b> exhibited antibacterial activities with minimum inhibitory concentration values of 2-8 μg/mL against the tested bacteria, which are superior to those of the reported dual-site ThrRS inhibitors. Our study provides the first proof-of-concept for developing triple-site inhibitors against aaRSs, inspiring future aaRS-based drug discoveries.