A comprehensive study on essential oils (EOs) extracted from some Mentha suaveolens L. samples, collected in the countryside of Tarquinia, is reported. In this study, the procedure for essential oil preparation, in terms of harvesting and extraction time, was analyzed in detail for the first time. The GC/MS analysis, carried out on 18 samples, revealed that piperitenone oxide (PO), the main essential oils’ chemical constituent, is primarily responsible for the related antifungal activity. Nevertheless, EOs with lower PO content indicate that other chemicals, such as para-cymenene, may participate in exerting the EOs’ antifungal effect. Furthermore, the bacterial reverse mutation assay highlighted lack of mutagenic effect in all tested samples. Analysis of the results indicated that for higher activity, the essential oils should be produced with 3 h maximum hydrodistillation, regardless of the harvesting time. Differently, the maximum essential oil yield can be obtained in August and the highest piperitenone oxide percentage is obtainable in July.