High temperature is a major threat toPleurotus ostreatuscultivation. In this study, a potential mechanism by whichP. ostreatusmycelia growth is inhibited under heat stress was explored. Lactate, as a microbial fermentation product, was found unexpectedly in the mycelia ofP. ostreatusunder heat stress, and the time-dependent accumulation and corresponding inhibitory effect of lactate on mycelial growth was further confirmed. The addition of a glycolysis inhibitor,2-deoxy-d-glucose (2DG), reduced the lactate content in mycelia and slightly restored mycelial growth under high-temperature conditions, which indicated the accumulation of lactate can be inhibited by glycolysis inhibition. Further data revealed mitochondrial dysfunction under high-temperature conditions, with evidence of decreased oxygen consumption and adenosine triphosphate (ATP) synthesis and increased reactive oxygen species (ROS). The removal of ROS with ascorbic acid decreased the lactate content, and mycelial growth recovered to a certain extent, indicating lactate accumulation could be affected by the mitochondrial ROS. Moreover, metabolic data showed that glycolysis and the tricarboxylic acid cycle were enhanced. This study reported the accumulation of lactate inP. ostreatusmycelia under heat stress and the inhibitory effect of lactate on the growth of mycelia, which might provide further insights into the stress response mechanism of edible fungi.