Many studies have demonstrated the herbicidal effects of several essential oils and their possible use as substitutes for chemical herbicides. Several enzymes play a very significant role in seed germination: among these, α-amylase could be involved in essential oil phytotoxic processes. The aims of this study were to compare the chemical composition of the essential oils of two ecotypes of O. heracleoticum growing in Cilento (Southern Italy) and to study their possible use as natural herbicide using Raphanus sativus, Sinapis arvensis and Lolium multiflorum seeds. Moreover, a possible inhibitory activity on the α-amylase enzyme extracted from germinating seeds was evaluated as a possible mechanism of action. Both oils, characterized by GC-MS, belonged to a carvacrol chemotype. The alpha-amylase activity was determined using DNSA (dinitrosalicylic acid) assay quantifying the reducing sugar produced. Furthermore, the essential oils demonstrated phytotoxicity at the highest dose tested, and an inhibitory effect on α-amylase, probably correlated with the phytotoxic effects, was registered. The oils showed interesting phytotoxic and alpha-amylase inhibitory activities, which deserve to be further investigated.