Sesquiterpene lactones (STLs) are important secondary metabolites synthesized in the aerial parts of Arnica montana L. The accumulation of STLs in micropropagated shoots treated with yeast extract (YE), salicylic acid (SA) and methyl jasmonate (MeJA) was measured by HPLC, while qualitative analysis was performed using HPLC-DAD-ESI-MS. YE (100 mg/L) and SA (200 μM) applied separately increased the total STL content by nearly 4.5-fold and 3-fold, respectively, compared to the control. The shoots treated with MeJA displayed the least pronounced changes in STL levels. The RT-qPCR analysis revealed a similar pattern of expression in both YE- and SA-treated shoots, characterized by strong upregulation of key genes (GAS and GAO) involved in the final steps of STL biosynthesis. YE elicitation resulted also in increased transcription of FDS, which provides the universal C15 precursor for sesquiterpenes. SA treatment led to the induction of HMGCR—encoding a key rate-limiting enzyme in the mevalonate pathway. Although MeJA also led to upregulation of both HMGCR and FDS, the effect of this elicitor was minimal. None of the treatments affected the expression of genes associated with the MEP/DOXP pathway (DXS and DXR). These findings provide valuable insights for future investigations into molecular mechanisms regulating STLs biosynthesis in A. montana.