Escherichia coli and Aspergillus fumigatus are two pathogens largely present among poultry. They can cause mild or severe forms of disease, and are associated with significant economic losses. The aim of the present study was to investigate the chemical composition and the in vitro antimicrobial activity of sixteen essential oils (EOs) and five mixtures against E. coli and A. fumigatus strains previously isolated from poultry. The study was performed with the following EOs: Aloysia
tryphilla, Boswellia
sacra, Cinnamomum zeylanicum, Citrus aurantium, Citrus bergamia, Citrus limon, Citrus reticulata, Cymbopogon citratus, Eucalyptus globulus, Lavandula hybrida, Litsea cubeba, Ocimum basilicum, Melaleuca alternifolia, Mentha piperita, Pelargonium
graveolens, and Syzygium aromaticum. Moreover, the following mixtures were also tested: L. cubeba and C. citratus (M1), L. cubeba and A. triphylla (M2), A. triphylla and C. citratus (M3), A. triphylla, C.
citratus and L. cubeba (M4), S. aromaticum and C. zeylanicum (M5). One hundred and ninety-one compounds were identified in the tested EOs and mixtures. MIC determination found good anti-E. coli activity with C. zeylanicum (2.52 mg/mL), C. citratus (1.118 mg/mL), L. cubeba (1.106 mg/mL), M. piperita (1.14 mg/mL) and S. aromaticum (1.318 mg/mL) EOs. Among the mixtures, M5 showed the best result with a MIC value of 2.578 mg/mL. The best antimycotic activity was showed by A. triphylla (0.855 mg/mL), followed by C. citratus (0.895 mg/mL), while C. aurantium, M. piperita, B. sacra and P. graveolens did not yield any antifungal effect at the highest dilution. The mixtures exhibited no antifungal activity at all. This study shows promising results in order to use EOs in the environment for disinfection purposes in poultry farms and/or in hatcheries.