The enzyme aspartate β-semialdehyde dehydrogenase (ASADH) plays a vital role in biosynthesis of essential amino acids and several important metabolites in microbes and some higher plants. So this key enzyme can be targeted selectively in these microbes to exhibit anti-bacterial and fungicidal effects. In this work, molecular modeling and comparative active site binding mode studies were performed for understanding the mode of action, in silico insight into the 3D structure, enzyme-substrate interactions with natural substrate in this homologous enzyme family. During comparative sequence analysis, high diversity was found in the sequences of different ASADHs and exhibited the same key binding interactions with the substrate. Both, the functional carboxylic and the phosphate group of the substrate are engaged in a bidentate interaction with the guanidinium N atom of two key arginyl active site residues of ASADHs. These structural and active site binding mode characterization studies can further be used for designing the more potent and selective substrate analogues inhibitors against ASADH family.