<p><i>Rubus fraxinifolius</i> Poir. is a wild raspberry species with promising medicinal potential, although its utilization is limited by low biomass availability in natural habitats. Elicitation is an effective strategy for enhancing biomass production and secondary metabolite accumulation in in vitro cultures, thereby improving the medicinal and nutritional value of plant-derived materials. This study investigated the effects of biotic elicitation on callus growth, biomass accumulation, secondary metabolite production, and antioxidant activity in <i>R. fraxinifolius</i> callus cultures. Four-week-old calli induced from 8-week-old in vitro seedlings on MS medium were cultured on MS medium supplemented with 0–100 mg L^-1 YE for 56 days, with one subculture performed at week 4. Morphological characteristics, fresh and dry weights, TPC, TFC, and DPPH radical scavenging activity were evaluated, followed by GC–MS profiling. Treatment of YE did not alter callus texture, which remained compact and dense across treatments, however, it significantly influenced growth and metabolic responses. The highest TPC (26.48&#xa0;mg GAE g^-1 DW) was recorded at 50 mg L^-1 YE, and the highest TFC (20.98&#xa0;mg QE g^-1 DW) occurred at 25 mg L^-1 YE. PCA and correlation analysis revealed that moderate YE concentrations (25–50 mg L^-1) were positively associated with phenolic and flavonoid accumulation, while higher concentrations were more closely related to biomass traits. TPC and TFC showed strong negative correlations with IC₅₀ values, indicating enhanced antioxidant activity. GC–MS analysis revealed increased tocopherol and squalene levels in YE-elicited <i>R. fraxinifolius</i> calli, confirming the potential of elicitation for enhancing bioactive metabolite production.</p>