Carlina acaulis L. (Asteraceae) is a medicinal plant with a long history of use as an herbal remedy. Recently, this species attracted the interest of agrochemical companies due to the presence of valuable biopesticides among which the most promising is carlina oxide (COx). To stimulate the biosynthesis of its specialised metabolites by elicitation, a polysaccharide – chitosan was supplemented as chitosan lactate (ChL). The aim was to evaluate the effects of ChL soil and foliar application on the C. acaulis biomass, photosynthetic efficiency, and levels of the COx, chlorogenic acids, protocatechuic acid, schaftosides, and triterpenic acids. ChL has been shown to have no effect on shoot growth while considerably increasing root biomass. It improved photosynthetic efficiency and enhanced levels of specialised metabolites. Soil application of ChL significantly increased chlorogenic and neochlorogenic acid content in leaves, by approximately 330% and 70%, respectively compared to controls. In contrast, foliar application more strongly increased COx levels in roots (55% for foliar vs. 16% for soil treatment). This study has demonstrated that the application of ChL can be used as an inexpensive and accessible way to boost the production of valuable compounds in C. acaulis.