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 Salmonella enterica threonyl-tRNA synthetase (SeThrRS). Competition of our compounds with all three substrates on SeThrRS binding was confirmed via isothermal titration calorimetry assays. Cocrystal structures of three compounds bound to SeThrRS unambiguously confirmed their substrate-mimicking triple-site binding mode. Compound 36j exhibited the best enzyme activity against SeThrRS with IC50 = 19 nM and Kd = 35.4 nM. Compounds 36b, 36k, and 36l 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.