1-Octen-3-ol is considered a major contributor to the characteristic mushroom aroma. It has been proposed that in the oyster mushroom Pleurotus ostreatus, 1-octen-3-ol is synthesized through two pathways comprising either 13-hydroperoxy-9Z,11E-octadecadienoic acid (13-HPOD) or 10-hydroperoxy-8E,12Z-octadecadienoic acid as an intermediate, and that 13-HPOD is produced by the oxidation of linoleic acid by lipoxygenase (LOX). However, information on 1-octen-3-ol in this mushroom remains limited. This study investigated variations in 1-octen-3-ol and the LOX gene (Polox1 and Polox2) expression together with LOX activity in P. ostreatus, according to fruiting body development, tissue specificity, maturity, and postharvest storage. 1-Octen-3-ol content was abundant in the stipe of medium-sized fruiting bodies (40–50 mm cap diam), but decreased after 2 d of storage at 4 °C. During fruiting body development, changes in LOX activity mimicked changes in transcript levels of Polox1, which was much more abundantly expressed than Polox2. The changes in LOX activity were different from changes in 1-octen-3-ol levels according to fruiting body development, maturity, and postharvest storage. Our investigation suggests that medium-sized fruiting bodies that were not subjected to long-term storage would have a good aroma quality and that there is no correlation between 1-octen-3-ol synthesis and LOX activity, which is largely dependent on Polox1 expression.