<p><i>Achillea millefolium</i> (Yarrow: Asteraceae family) is found on various continents worldwide. Yarrow is utilized as a medicinal plant with numerous pharmacological effects on the human body. In vitro plant culture is a novel approach to obtaining active metabolites from this plant. To enhance the pharmacologically active metabolites and morphological characteristics of this plant, certain substances have been suggested for addition to the culture medium. Due to the role of zirconium oxide (ZrO₂) in plant metabolism and growth, this substance can serve as an elicitor to influence the production of certain plant secondary metabolites. This study aimed to explore the impact of zirconium oxide on the secondary metabolites of <i>Achillea millefolium</i> through in vitro culture. First, thirty-day-old seedlings were treated with the elicitor for one month. Subsequently, secondary metabolites were analyzed using LC-MS and GC-MS methods. Furthermore, the growth parameters, total phenolic contents, antioxidant properties, photosynthetic pigments, volatile and phenolic compounds of their extracts were examined. In growth parameters, only the fresh weight (20 ppm) and dry weight (10 ppm) of (ZrO₂) seedlings increased compared to the control, respectively (P-value &lt; 0.05). Concerning the volatile contents, non-terpenoids were found to be more significant than terpenoids in all treatments. It is important to note that some essential compounds were generated only in the presence of ZrO₂. Furthermore, the GC-MS results indicated a higher number of volatile compounds in the control treatment compared to other treatments, potentially due to the greater number of trichomes in the control group. The LC-MS findings revealed a gentle increase in phenolic compounds, particularly caffeic acid.</p>