Significance Microbial resistance to β-lactam antibiotics mediated by β-lactamase–catalyzed hydrolysis is a major global health concern. Penam sulfones, which are structurally related to penicillins, inhibit clinically important serine β-lactamases (SBLs) by forming transiently stable covalent complexes, thereby protecting β-lactam antibiotics from hydrolysis. The characterization of these complexes and mechanisms of SBL inhibition is important for development of new SBL inhibitors (SBLi). Studies on the mechanism of the new SBLi enmetazobactam employing mass spectrometry and X-ray crystallography inform on its mode of action and also lead to reevaluation of mechanisms of current clinically important SBLi. In addition to insights into the mechanisms of transient SBL inhibition by penam sulfones, the results reveal potential for penam sulfone optimization to enable irreversible SBL inhibition.