In this study, responses of carboxylesterases, acetylcholinesterase, and stress protein Hsp70 were examined in the midgut and midgut tissue, and brain of fifth instar larvae of Lymantria dispar L. and Euproctis chrysorrhoea L. following chronic exposure to dietary fluoranthene. Specific carboxylesterase activity increased significantly in the midgut tissue of E. chrysorrhoea larvae treated with a lower fluoranthene concentration. The specific patterns of isoforms expression, recorded in larvae of both species, enable efficient carboxylesterase activity as a significant part of defense mechanisms. Increased Hsp70 concentration in the brain of L. dispar larvae points to a response to the proteotoxic effects of a lower fluoranthene concentration. Decreased Hsp70 in the brain of E. chrysorrhoea larvae in both treated groups can suggest induction of other mechanisms of defense. The results indicate the importance of the examined parameters in larvae of both species exposed to the pollutant, as well as their potential as biomarkers. •Exposure to dietary fluoranthene led to prolonged development of L. dispar and E. chrysorrhoea larvae.•Specific CarEs activity increased in the midgut tissue of E. chrysorrhoea larvae exposed to dietary fluoranthene.•Species-specific expression patterns of CarEs isoforms were recorded in larval midguts.•Differences in Hsp70 responses were revealed in larval brain tissues of examined species.