Medicinal plants produce several natural bioactive molecules and compounds called secondary metabolites that have very important biological properties. The objective of this work is to determine the chemical composition of essential oils (EO) obtained from the leaves of three Tunisian species of Eucalyptus (E. oleosa, E. pimpiniana, E. polyanthemos) and to evaluate their biological activities. As a result, 45 different compounds were identified: 26 from E. pimpiniana, 15 from E. polyanthemos, and 39 from E. oleosa, which represent 99.4%, 99.4% and 98.6%, respectively, of the entire essential oil constituents. The analyses showed that 1,8-cineole (35.3%) and β-eudesmol (25.5%) were the main components in E. pimpiniana essential oil, whereas 1,8-cineole (71.6%) and globulol (13.2%) characterized E. polyanthemos leaf oil. Also, 1,8-cineole (13.4%), spathulenol (11.9%), and β-eudesmol (8.5%) were found to be the main constituents of E. oleosa EO. Other compounds, such as phellandral, p-cymen-7-ol (syn. cumin alcohol), carvacrol, myrtenal, cumin aldehyde and cryptone, are specific to the EOs of E. oleosa, making it distinct from the other Eucalyptus species studied. The essential oils showed low antioxidant capacity, but significant antifungal activity against five Fusarium spp. Indeed, E. oleosa essential oil exhibited the highest level of antifungal activity. Additionally, herbicidal activity has only been proved in a preliminary in vitro test against 3 weed species (Sinapis arvensis L., and Lepidium sativum) of the same family. The greatest inhibition of seed germination was obtained with the E. oleosa essential oils even at low concentrations strongly suggesting that they could have application in agriculture, particularly as antagonists against Fusarium and other fungi and for weed control.