Natural defensive substances synthesized by plants that could replace synthetic pesticides in the protection of plants against insect invasions are constantly being sought. The study assessed changes in the qualitative and quantitative composition of secondary metabolites in horse chestnut leaves collected in different locations and differing in the sensitivity of the plant to the invasion by the horse-chestnut leaf miner. An attempt was made to identify compounds that are most responsible for the increased plant resistance to this threat. Additionally, changes in the anatomy of chestnut leaves affected by the pest were presented. It was noticed that the trees differed in the composition of secondary metabolites already in the initial growing season, which should be related to the influence of habitat conditions. The analysis of the profile of the compounds in non-infested and infested horse chestnut leaves revealed a clear response of the plant to the stress factor, i.e., the foraging of the horse-chestnut leaf miner. Catechins seem to be compounds involved in plant resistance. The leaf anatomy showed enhanced accumulation of phenolic compounds at the pest foraging sites. Hypertrophy and thickened and cracked cell walls of the spongy parenchyma were visible in the vicinity of the mines.