<b>Chemical and Enantiomeric Profiling, Chemotype Classification and Antibacterial Activity of a Phytone-Rich Essential Oil from </b><b><i>Lawsonia inermis</i></b><b> Leaves</b>

Purpose: This study examines the chemical composition, enantiomeric distribution, and antibacterial activity of Lawsonia inermis leaf essential oil to identify its chemotypic features and clarify links between composition and bioactivity. Methodology: Essential oil was obtained from L. inermis leaves via hydro-distillation. Chemical characterization was performed using GC–MS, while chiral GC–MS was used to determine the enantiomeric distribution of selected terpenoids. Hierarchical cluster analysis (HCA) compared the oil with previously reported L. inermis chemotypes. Antibacterial activity of the crude oil and selected constituents (phytone, linalool, and trans-α-farnesene) was evaluated against seven ATCC bacterial strains using the microbroth dilution method. Results: A total of 87 compounds were identified, representing 97.1% of the essential oil. The dominant constituents were phytone (15.4%), palmitic acid (14.6%), and tetradecanoic acid (5.5%). Enantiomeric profiling revealed several non-racemic terpenoids, including α-terpineol, sabinene, β-pinene, and trans-nerolidol, each exhibiting high enantiomeric excesses (>70%). HCA positioned the Nigerian oil within cineole- and linalool-related chemotypes, while also revealing a distinctive phytone-dominant profile. Antibacterial assays showed moderate activity of the crude oil (MIC 1250–5000 μg/mL), whereas isolated constituents particularly phytone and linalool demonstrated stronger antibacterial effects (MIC 78.1–625 μg/mL). Novelty and Contribution: This study reports, for the first time, the enantiomeric distribution of L. inermis leaf essential oil and identifies a novel phytone-rich chemotype. Practical and Social Implications: The findings support improved quality assessment and standardization of L. inermis essential oil and highlight its potential for developing plant-based antibacterial agents.