A recent report discovered a chloro-hydantoin lead for COVID-19 antiviral therapy, however, effects of stereochemical changes of the spiro-cycle and halogens were not examined. Herein, we describe how minimal structural variations, such as stereochemistry and atomic size of halogen substituents, affect binding affinity and biological activity. Blind docking derived a correlation between binding affinity and IC50 of various hydantoin analogs revealed the F compound 8 (2s, 4s) to be more potent than Cl compound 9a (2s, 4s) and Br compound 10a (2s, 4s). However, experimentally, Cl compound 9a and Br compound 10a exhibited greater in vitro potency against Mpro than F compound 8. To rationalize the impact of atomic size and stereo configuration on observed biological activity, the volume occupancy and protein-ligand interactions were evaluated. Compounds 9a and 10a occupy 33% and 35% of the cavity space. The Br atom’s size optimally fits the cavity with less conformational flexibility, conferring higher stability. MD simulation data of the ligand torsion profile and the solvent accessible surface area also indicated higher conformational stability for compound 10a than compounds 8 and 9a. Compounds 9b and 10b, (2r, 4r) stereoisomers of 9a and 10a, had more conformational flexibility and were less active Mpro inhibitors in vitro.