| Summary: | The <i>Eucalyptus</i> genus (Myrtaceae) is characterized by a richness in essential oils (EO) with multiple biological activities. This study reports the chemical composition and the phytotoxic and antimicrobial activities of the EOs from Tunisian <i>E.</i><i>occidentalis</i>, <i>E.</i><i>striaticalyx</i> and <i>E.</i><i>stricklandii</i>. The EOs were analyzed using GC/MS and their phytotoxicities were assessed against the germination and seedling growth of <i>Sinapis arvensis</i>, <i>Trifolium campestre</i> and <i>Lolium rigidum</i>. Antimicrobial activity was investigated against both Gram-negative (<i>Pseudomonas aeruginosa, Escherichia coli</i> and <i>Acinetobacter baumannii)</i> and Gram-positive (<i>Staphylococcus aureus</i> and <i>Listeria monocytogenes</i>) bacteria. The inhibition of biofilm formation and its metabolism was determined at different times. All EOs were rich in oxygenated monoterpenes (36.3–84.8%); the EO of <i>E.</i><i>occidentalis</i> was rich in sesquiterpenes, both oxygenated and hydrocarbon (40.0% and 15.0%, respectively). Eucalyptol was the main constituent in all samples. The EOs showed phytotoxic activity on seed germination and seedling growth, depending both on chemical composition and weed. The EOs show a remarkable antibacterial potential resulting in a significant inhibition of the formation of bacterial biofilm and its metabolism, depending on the EO and the strain, with activity on the mature biofilm as well. Therefore, these <i>Eucalyptus</i> EOs could have potential applications both in the food and health fields.
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