Thymus sp. (L.) encompasses over 500 aromatic species distributed in the Mediterranean area, in dry, rocky environments up to 1800 m above sea level. Plants of Thymus sp. (L.) from the Serpyllum group, growing in five areas of the Pistoia Mountains, were georeferenced and sampled for a chemical characterization of the terpene content. Fresh biomass was collected to distill thyme essential oil, and the derived hydrolate was recovered as byproduct of the process. The antifungal efficacy of the chemically characterized thyme essential oil and hydrolate was evaluated on two fungal biodeteriogens typically affecting paper objects, Alternaria alternata and Cladosporium cladosporioides. High thymol content thyme essential oil (about 75 %) and hydrolate (about 62 %) were obtained from steam distillation and used to perform in vitro tests on paper samples following a non-invasive approach. For both fungi, a significant growth reduction on inoculated Whatman paper was observed with both hydrolate and thyme essential oil. The GC-MS analysis of 94 thyme samples led to the characterization of seven chemotype classes, and a PCA was carried out to investigate the potential impact of different collection sites on the chemotypes variability. These findings highlight the necessity of an accurate selection of the plant material for an optimized production of thyme essential oil and hydrolate with improved antifungal efficacy.

The Pistoia Mountains exhibit a great variety of flora, particularly rich in aromatic plants, such as juniper, mint, savory, helichrysum, and thyme. Thyme is especially notable for its essential oil, typically displaying high thymol and carvacrol content. While the chemotype of thyme determined by its specific terpene composition is genetically controlled, environmental factors, plant age, and seasonality can influence terpene production. This article investigates the morpho-chemical variability of wild thyme plants collected from two different regions of the Pistoia Mountains, identifying five distinct chemotypes. The gas chromatography–mass spectrometry (GC-MS) technique was used to characterize the chemical profiles and determine the seasonal variation in terpene production, identifying spring and summer as the balsamic period, the optimal time for essential oil collection. Furthermore, high-value thyme clones were preserved through in vitro micropropagation, ensuring chemotype stability. These findings contribute to a deeper understanding of wild thyme biodiversity and provide a foundation for practical applications, including the development of value-added products like herb-infused cheeses, plant and animal disease treatments, and integrated pest management strategies in agricultural systems.