Melissa officinalis L. is a well-known herb for its ethnopharmacological properties owing to its essential oils. However, essential oil yield remains relatively low when compared to other members of the Lamiaceae family, necessitating breeding for improvement. This study investigated oryzalin as an antimitotic agent to induce polyploidy in M. officinalis for crop improvement mainly aiming for higher essential oil yield. Nodal segments were micropropagated and exposed to 20, 40, and 60 μM oryzalin for 24 and 48 hours. The survival rate declined exponentially with higher concentrations and durations. The highest polyploid induction rate (8%) occurred with 40 μM for 24 hours. Tetraploid plants displayed vigorous growth, with longer shoots, larger leaves, and more leaves per shoot. Their average essential oil yield increased by 75% due to larger peltate trichomes. Tetraploid and diploid plants shared geranial, neral, and citronellal as major components although 11.06% and 9.49% increase in geranial and neral, respectively was observed in tetraploid plants. In conclusion, oryzalin effectively induced polyploidy in M. officinalis, leading to tetraploid plants with superior agronomical traits and significantly higher essential oil yield. These findings hold promising avenue for meeting commercial demand and advancing the cultivation of this valuable herb. Further research on optimizing polyploid induction methods could contribute to the growth of Melissa officinalis L. as a more productive and economically viable crop.