β-Lactam resistance of methicillin-resistant Staphylococcus aureus (MRSA), a pathogenic bacterium that causes staph infections, represents a serious threat to public health. This arises primarily due to the inability of β-lactam antibiotics to inhibit the transpeptidase activity of penicillin-binding protein 2a (PBP2a). Effective inhibition of PBP2a to prevent the bacterial cell wall biosynthesis is of great importance for the treatment of a variety of clinically challenging infectious diseases caused by MRSA. To gain fundamental insights into the mode of covalent inhibition of the enzyme, we have carried out computational studies of the acylation reactions between small β-lactam molecules (methicilin and nitrocefin) and PBP2a using the B3LYP/6-31G* and ONIOM(B3LYP/6-31G*:AMBER) hybrid quantum mechanical/molecular mechanical methods. Our calculations show that the acylation involves two transition states and that methicilin and nitrocefin undergo acylation in slightly different manners. The acylation of nitrocefin is more facile, which is attributed to the larger release of ring strain and the larger resonance stabilization gained upon ring opening. We suggest that, in addition to the nonbonded interactions between the ligand and the protein, these quantum chemical factors, which are associated with efficiency of the acylation step, should be taken into account and carefully controlled in designing novel β-lactam inhibitors of PBP2a.