Plant's essential oils have a wide range of allelopathic effects with potential uses as bioherbicides. In addition, the application of oils through nanoemulsions represents a promising alternative for agriculture, as it offers better performance and lowers toxic waste generation. Therefore, this study aimed to evaluate the chemical constitution of Rosmarinus officinalis (rosemary) essential oil, as well as its effects on germination, initial growth, Catalase (CAT), Peroxidase (POX), and Superoxide-Dismutase (SOD) enzymes activity of Lactuca sativa seeds. Nanoemulsions were produced at 5% concentration and then diluted with distilled water to 5.0, 7.0, and 10.0 mg/mL. We compared data obtained using variance (ANOVA) analysis, followed by Tukey's test at 5% probability. Rosemary oil showed a wide variety of terpenoid compounds, mainly the 1,8-cineol monoterpene, which accounted for 46% of the sample. The oil showed a dose-dependent negative allelopathic influence on all variables analyzed, causing a drop in germination percentage (%G), germination speed index (GVI), mean germination time (MTG), and leaf and root length. There was no change in CAT and SOD activity. The POX activity showed a reduction starting at the concentration of 7.0 mg/mL. The results showed allelopathic effects of rosemary oil, with potential use as a natural bioherbicide.