The enzymic oxidation of the polyunsaturated fatty acid-linoleic acid leads, in fungi, to the formation of a unique class of nonconjugated hydroperoxides, which are cleaved to form eight-carbon volatiles characteristic of mushroom and fungal flavor. However, the enzymes involved in this biosynthetic pathway, the bioavailability of the fatty acid substrate, and the occurrence of the reaction products (hydroperoxides and eight-carbon volatiles) are not fully understood. This study investigated the lipids, fatty acids, and hydroperoxide levels, as well as eight-carbon volatile variations in the fungal model Agaricus bisporus, according to four parameters: sporophore development, postharvest storage, tissue type, and damage. Eight-carbon volatiles were measured using solid phase microextraction and gas chromatography-mass spectrometry. Tissue disruption had a major impact on the volatile profile, both qualitatively and quantitatively; 3-octanone was identified as the main eight-carbon volatile in whole and sliced sporophore, an observation overlooked in previous studies due to the use of tissue disruption and solvent extraction for analysis. Fatty acid oxidation and eight-carbon volatile emissions decreased with sporophore development and storage, and differed according to tissue type. The release of 1-octen-3-ol and 3-octanone by incubation of sporophore tissue homogenate with free linoleic acid was inhibited by acetylsalicylic acid, providing evidence for the involvement of a heme-dioxygenase in eight-carbon volatile production.