Essential oils (EOs) have been extensively studied as valuable eco-friendly compounds with herbicidal activity for weed management. Phytotoxic potential of EOs, extracted from a wild population of Origanum vulgare ssp. hirtum (Link) Ietswaart, has been here evaluated on plant model Arabidopsis, through a physiological and metabolomic approach. The EOs composition was mainly characterized by monoterpenes and sesquiterpenes, with a strong abundance of two monoterpenic phenols, namely carvacrol and thymol, and the monoterpene o-cymene. The in vitro bioassay confirmed a strong phytotoxic effect of EOs on Arabidopsis rosettes, showing by both a strong growth reduction and highly chlorotic leaves. In well-developed seedlings, EOs firstly caused growth reduction and leaf chlorosis, together with a series of interconnected metabolic alterations: i) impairing the nitrogen assimilation into amino acids, which affects in particular the glutamine metabolism; and as consequence ii) excessive accumulation of toxic ammonia into the leaves, associated with oxidative stress and damage; iii) declining the efficiency of the photosynthetic apparatus, connected to the reduced CO2 fixation and photooxidation protection; iv) impairing the photorespiratory pathway. Overall, the results highlights that EOs alters principally the ability of Arabidopsis seedlings to incorporate inorganic nitrogen into amino acids, principally glutamine, leading to a dramatic accumulation of ammonia in leaf cells. This primary effect induces, in turn, a cascade of reactions that limits the efficiency of PSII, inducing oxidative stress and finally causing a strong plant growth reduction, leaf necrosis and eventually plant death. These findings suggest that O. vulgare EOs might be proficiently exploited as a potential bioherbicide in an ecofriendly agriculture. Moreover, its multitarget activity could be advantageous in limiting weed resistance phenomenon.