Bombardier beetles are well-known for their remarkable defensive mechanism. Their defensive apparatus consists of two compartments known as the reservoir and the reaction chamber. When challenged, muscles surrounding the reservoir contract sending chemical precursors into the reaction chamber where they mix with enzymes resulting in an explosive discharge of a hot noxious chemical spray containing two major quinones: 1,4-benzoquinone and 2-methyl-1,4-benzoquinone (toluquinone). Previously, it has been speculated that the biosynthesis of all benzoquinones originates from one core precursor, 1,4-hydroquinone. Careful ligation of the base of the reservoir chamber enabled us to prevent the explosive reaction and sample untransformed reservoir fluid, which showed that it accumulates significant quantities of 1,4-hydroquinone and 2-methyl-1,4-hydroquinone. We investigated the biosynthetic mechanisms leading to quinone formation by injecting or feeding Brachinus elongatulus beetles with stable-isotope-labeled precursors. Chemical analysis of defensive secretion samples obtained from 1,4-hydroquinone-d6-administered beetles demonstrated that it underwent conversion specifically to 1,4-benzoquinone. Analogously, results from m-cresol-d8 injected or fed beetles confirmed that m-cresol is metabolized to 2-methyl-1,4-hydroquinone, which is then oxidized to 2-methyl-1,4-benzoquinone in the hot spray. Our results refute the previous claim that 1,4-hydroquinone is the precursor of all substituted benzoquinones in bombardier beetles and reveal that they are biosynthetic products of two independent pathways. Most likely, the aforementioned biosynthetic channel of hydroxylation of appropriate phenolic precursors and subsequent oxidation is not restricted to bombardier beetles; it could well be a general pathway that leads to the formation of all congeners of benzoquinones, one of the most widely distributed groups of defensive compounds in arthropods.