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The essential oil (EO) of Thymbra capitata has been demonstrated to possess herbicidal activity and could be used as an alternative to synthetic herbicides with reduced persistence in soil and new mode of action. Nevertheless, it is necessary to determine the adequate doses for its use, the proper way for its application and the best phenological stage of weeds and crops in which the EO should be applied to obtain maximum efficacy against weeds without compromising crop production. In this work, T. capitata EO was tested at three different concentrations against weeds grown from a citrus orchard soil seedbank untreated with herbicides and against three important weed species grown in substrate to determine the efficacy of the concentrations on different weed species. All experiments were carried out under greenhouse conditions. To find out the best way for applying the EO, it was applied by irrigation and by spraying on the targeted weeds, and to verify the influence of timing, it was tested on Lolium rigidum at two different phenological stages and on wheat at a later phenological stage than weeds. The highest concentration tested (12 µL·mL−1) showed the best performance to control weeds. The more effective mode of application was by spraying on dicotyledons and by irrigation on monocotyledons at the earliest phenological stage. T. capitata EO was phytotoxic for wheat. More trials in different crops are needed to determine the best conditions for its use. [ABSTRACT FROM AUTHOR]