The aim of the present study was to elucidate the effect of the food additive tert-butylhydroquinone (TBHQ), its metabolite tert-butylbenzoquinone (TBQ), the indirect food additive 2,5-di-tert-butylhydroquinone (DTBHQ) and five other structurally related quinones on the enzymatic activity of cyclooxygenase-1 and -2 (COX-1, -2) and 5-lipoxygenase (5-LO). In a study conducted on purified enzymes TBHQ affected neither COX-1 nor COX-2 activity, but inhibited 5-LO in human neutrophils with an IC50 of 13.5 µM. In contrast, DTBHQ regulated the activity of 5-LO as well as the activity of COX-2 (IC50 = 1.8 and 14.1 for 5-LO and COX-2, respectively). The in silico studies on DTBHQ indicated interactions with the catalytic iron of 5-LO and possible hydrogen bond interactions with His367 or His372. These data suggest that TBHQ and DTBHQ influence arachidonic acid (AA) metabolism in biologically relevant concentrations in vitro and should be further studied for their possible effect on the AA pathway in vivo.