Botulinum neurotoxins have remarkable persistence (~weeks to months in cells), outlasting the small molecule inhibitors designed to target them. To address this disconnect, inhibitors bearing two pharmacophores-a zinc binding group and a Cys-reactive warhead-were designed to leverage both affinity and reactivity. A series of 1 st generation bifunctional inhibitors was achieved through structure-based inhibitor design. Through X-ray crystallography, engagement of both the catalytic Zn 2+ and Cys165 was confirmed. A 2 nd generation series improved on affinity by incorporating known reversible inhibitor pharmacophores; the mechanism was confirmed by exhaustive dialysis, mass spectrometry, and in vitro evaluation against the C165S mutant. Finally, a 3 rd generation inhibitor was shown to have good cellular activity and low toxicity. In addition to our findings, an alternative method of modeling time-dependent inhibition that simplifies assay setup and allows comparison of inhibition models is discussed.