The phosphotriesterase (PTE) activity of a series of binuclear and mononuclear zinc(II) complexes and metallo-β-lactamase (mβl) from Bacillus cereus was studied. The binuclear complex 1, which exhibits good mβl activity, shows poor PTE activity. In contrast, complex 2, a poor mimic of mβl, exhibits much higher activity than 1. The replacement of Cl− ligands by OH− is important for the high PTE activity of complex 2 because this complex does not show any catalytic activity in methanol. The natural enzyme mβl from B. cereus is also found to be an inefficient catalyst in the hydrolysis of phosphotriesters. These observations indicate that the binding of β-lactam substrates at the binuclear zinc(II) center is different from that of phosphotriesters. Furthermore, phosphodiesters, the products from the hydrolysis of triesters, significantly inhibit the PTE activity of mβl and its functional mimics. Although the mononuclear complexes 3 and 4 exhibited significant mβl activity, these complexes are found to be almost inactive in the hydrolysis of phosphotriesters. These observations indicate that the elimination of phosphodiesters from the reaction site is important for the PTE activity of zinc(II) complexes.