Gram-positive bacterium Staphylococcus aureus (S. aureus) is one of the most common pathogens causing bovine mastitis, which is a worldwide problem in dairy herds. The emergence and spread of methicillin-resistant and other antibiotic-resistant strains warrants the need for developing inhibitors based on novel targets. Previous research has shown that bacterial threonyl-tRNA synthetase is a promising target for inhibitors development. In this study, a 3-dimensional structure of S. aureus threonyl-tRNA synthetase (SaThrRS) was obtained from RCSB Protein Data Bank (PDB ID: 1NYR). The ZINC natural product database including 11247 compounds was used for virtual screening against the ATP binding site of the target using AutoDock Vina program. The lead compounds were selected based on their affinities to the target. Since the binding free energy of selective inhibitor (borrelidin) was −9.3 kcal/mol, 173 compounds whose binding free energy was not greater than −10 kcal/mol were selected as potential inhibitors using excel in microsoft office program. The top 5 hits, with the binding free energy lower than −11 kcal/mol, are ZINC14778984 (−11.3 kcal/mol), ZINC67911489 (−11.1 kcal/mol), ZINC77269158 (−11.1 kcal/mol), ZINC33861464 (−11.0 kcal/mol), and ZINC77269178 (−11.0 kcal/mol). Among them, ZINC14778984 (−11.3 kcal/mol) was best lead because of its highest inhibitory activity. The binding site of ZINC14778984 on SaThrRS was analyzed using Discovery studio 2.5 program. And ZINC14778984 was located in a pocket consisted of 28 residues, which were MET334, ASN335, CYS336, MET363, ARG365, GLU367, LEU375, GLN376, ARG377, VAL378, MET381, LEU383, ASP385, SER386, HIS387, LYS471, ASP473, THR485, LEU486, THR488, GLN490, HIS517, ARG518, GLY519, SER522, THR523, GLU525, and ARG526. This study demonstrates the success of the screening method resulting in 5 novel SaThrRS inhibitors with potential for treating bovine mastitis.