Ragged Robin (Lychnis flos-cuculi L.) is a plant with notable medicinal potential, attributed to its wide spectrum of biological and pharmacological properties. Biotechnological methods were used for L. flos-cuculi to sustainably enhance the production of biomass and valuable secondary metabolites from a species with declining natural populations and promising pharmacological potential. The present study investigated the impact of biotechnological strategies—specifically, elicitation using Ganoderma lucidum (Reishi) extract and precursor feeding with L-phenylalanine (L-Phe)—on biomass growth and the accumulation of phenolic compounds in shoot cultures. These treatments were applied to clonally propagated, agitated shoot cultures. Chromatographic analyses confirmed the presence of species secondary metabolites and demonstrated that both elicitation and precursor feeding contributed to increased levels of key phenolic acids and flavonoids. Fungal extract elicitation and L-Phe supplementation enhanced the biosynthesis of several target compounds, with the effectiveness influenced by the duration of treatment and the concentration applied. Among the bioactive molecules stimulated were syringic acid, ferulic acid, caftaric acid, vitexin, and apigenin. Notably, precursor feeding with L-Phe promoted the accumulation of phenolic acids in a concentration-dependent manner and positively influenced flavonoid content, particularly vitexin and rutoside. These results support the application of elicitors and metabolic precursors as effective tools in plant biotechnology for optimizing the production of valuable secondary metabolites in L. flos-cuculi shoot cultures. Key message: This study demonstrated that in vitro shoot cultures of Ragged Robin can serve as a valuable source of phenolic compounds, particularly when enhanced through biotechnological treatments.