In this study, the essential oil of Acmella ciliata, characterized by the presence of significant amounts of spilanthol, α-phellandrene epoxide, and carvotanacetone, was assessed for its influence on the growth of Nicotiana tabacum cv. Xanthi, with the focus primarily on root hair density and primary root length. Following its extraction through microwave-assisted hydrodistillation, the oil was stored at 4°C in amber vials, distinguishable by its unique yellowish-reddish hue, with a refractive index of 1.3478 and a density of 0.847 g cm-3. Among the various dilutions evaluated, the undiluted oil (T3) and the dilution 1.5:0.5 (Oil:EtOH) (T6) demonstrated the most prominent effects. The T3 and T6 treatments markedly enhanced root hair numbers, with T6 additionally promoving root length compared to other treatments. Considering the presence of bioactive alkamides such as spilanthol in the oil, these compounds may have contributed to the observed root growth modulation. When compared against the positive control, affinin, Acmella ciliata essential oil displayed a more pronounced effect on root hair proliferation, while affinin predominantly boosted primary root elongation. The findings highlight the differential effects of the essential oil on specific plant growth parameters.