Essential oils (EOs) have been known for a long time, and they are used in several fields such as medicine and aromatherapy, as well as in the food and pharmaceutical industries. In the last decade, EOs have also been applied to contrast the biodeterioration of cultural heritage, representing a powerful resource in green conservation strategies. In this study, an integrated approach based on microscopic observation, in vitro culture, and molecular investigation was preliminarily employed to identify biological systems colonizing wooden artworks. In order to contrast the biodeterioration processes induced by fungal colonization (Aspergillus flavus) or insect infestation (Anobium punctatum), wooden artworks were exposed to the volatile compound of Origanum vulgare or Thymus vulgaris essential oils (EOs), the chemical composition of which was determined by GC–MS using both polar and apolar columns. Artwork exposure was performed in ad-hoc-assembled “clean chambers.” Evaluating the effects on biological systems, the compatibility with artwork constitutive materials, and the lack of negative effects on human health and environmental pollution, the use of EOs as a valid alternative to traditional biocides must be considered.