The inhibition mechanisms of the firefly luciferase (Luc) by three of the most important inhibitors of the reactions catalysed by Luc, dehydroluciferyl-coenzyme A (L-CoA), dehydroluciferin (L) and L-luciferin (L-LH2) were investigated. Light production in the presence and absence of these inhibitors (0.5 to 2 μM) has been measured in 50 mM Hepes buffer (pH = 7.5), 10 nM Luc, 250 μM ATP and D-luciferin (D-LH2, from 3.75 up to 120 μM). Nonlinear regression analysis with the appropriate kinetic models (Henri–Michaelis–Menten and William–Morrison equations) reveals that L-CoA is a non-competitive inhibitor of Luc (Ki = 0.88 ± 0.03 μM), L is a tight-binding uncompetitive inhibitor (Ki = 0.00490 ± 0.00009 μM) and L-LH2 acts as a mixed-type non-competitive-uncompetitive inhibitor (Ki = 0.68 ± 0.14 μM and αKi = 0.34 ± 0.16 μM). The Km values obtained for L-CoA, L and L-LH2 were 16.1 ± 1.0, 16.6 ± 2.3 and 14.4 ± 0.96 μM, respectively. L and L-LH2 are strong inhibitors of Luc, which may indicate an important role for these compounds in Luc characteristic flash profile. L-CoA Ki supports the conclusion that CoA can stimulate the light emission reaction by provoking the formation of a weaker inhibitor.