The aim of the study was to investigate how essential oil production and associated chemical composition and related biological activity could be influenced by different cultivation treatments and distillation methods. Foeniculum vulgare Mill. (fennel), Origanum vulgare L. (oregano) and Thymus vulgaris L. (thyme) were cultivated in absence of any fertilizer (control) and in presence of three different fertilizers: a chemical one with augmented mineral phosphorus and potassium, a second added with hydrolyzed organic substance and mineral phosphorus and potassium (organic–mineral) and a third one treated with a high content of organic nitrogen of protein origin (organic). The plants were subjected to steam distillation using two modalities, recycled and continuous, to obtain 32 essential oil samples. Chemical composition analysis was performed using gas chromatography–mass spectrometry; in vitro antimicrobial activity was evaluated using a broth microdilution method. In general, the recycled distillation method appeared to have a slightly higher yield than the continuous method. The “mineral” and “organic–mineral” treatments resulted in a higher yield compared to the “organic” or “control” treatments, and this was particularly evident in the recycled method. The “control” plants had a lower yield of essential oils. Anethole (13.9–59.5%) and estragole (13.4–52.2%) were the main constituents of the fennel oils; p-cymene and its derivatives carvacrol and thymol were the main constituents of the oregano and thyme samples. The antimicrobial activity of the thyme oils on Staphylococcus aureus ranged from 0.31 to 0.16% (v/v); a lower effect of the oregano samples and no activity of the fennel samples were observed. The essential oils failed to inhibit the growth of Pseudomonas aeruginosa strains.
