Microbial infections from pathogens remained a dire threat to mankind since time immemorial. Growing drug resistance is perpetually compounding the problem. Various multi-drug resistant bacterial, fungal and parasitic pathogenic strains continually pose elevated risk. Consequently, the necessity of developing new inhibitors is of tremendous importance. Growth of the pathogens can be inhibited by targeting the protein biosynthesis in the organism. Enzymes participating in the translation pathway such as aminoacyl tRNA synthetases (aaRSs) are promising targets for the development of new inhibitors. Detailed understanding of the interaction between the drug and the site where the drug binds (such as the active site or the editing site) is essential for a complete molecular level perspective of the inhibition of aaRSs in various pathogens. In the present chapter we depict the molecular studies of the inhibition of various aaRSs in bacterial, fungal and parasitic pathogens. The structural insights along with dynamical information about the inhibition processes of various class I and class II aaRSs are now developing. The studies include structural analysis based on crystallographic and NMR measurements. These studies are often complemented by mutation studies and kinetic methods. Molecular dynamics simulation and other computational studies provided detailed perspective of the interactions between inhibitor and the binding site. As a result, understanding of the actions of the various inhibitors of aaRSs emerged. The molecular level studies form the basis of new avenues for the development of novel inhibitors of bacterial, fungal and parasitic pathogens and hold promise for drug design in future.