The aminoacyl-tRNA synthetases are a family of enzymes involved in the process of translation, more specifically, ligating amino acids to their cognate tRNA molecules. Recent evidence suggests that aminoacyl-tRNA synthetases are capable of aminoacylating proteins, some of which are involved in the autophagy pathway. Here, we test the conditions under which E. coli and human threonyl-tRNA synthetases, as well as hisidyl-tRNA synthetase aminoacylate themselves. These reactions are ATP dependent, stimulated by Mg2+, and are inhibited by increasing cognate tRNA concentrations. These data represent the foundation for future aminoacylation experiments, specifically delving into the relationship between the autophagy pathway and the aminoacylation of proteins. Additionally, we provide evidence of the inhibitory abilities of the anti-bacterial [Beta]-lactone obafluorin on both E. coli and human threonyl-tRNA synthetases. Further, we also show that the benzoate obafluorin analog EHTS-1 significantly inhibits E. coli threonyl-tRNA synthetase but not the human enzyme. These data could be useful in determining the potential for obafluorin and EHTS-1 as anti-bacterial and possibly anti-angiogenic drugs.