Aromatic plants represent a source of natural products with medicinal properties, and are also utilized in the food and pharmaceutical industries. Recently, the need for eco-compatible and non-toxic products, safe for both the environment and human health, have been proposed for the sustainable conservation of historic–artistic artifacts. In this study, in order to counteract microbial colonization (Aspergillus sp., Streptomyces sp., Micrococcus sp.) on wooden artwork surfaces, Thymus vulgaris L. (Lamiaceae) essential oil (EO) and hydro-alcoholic (HA) solutions were applied in a polyphasic approach. The antimicrobial activities of EO and HA solutions were preliminarily assessed by agar disc diffusion (ADD) and well plate diffusion (WPD) in vitro methods, defining the specific concentration useful for bacterial and fungal genera, identified by optical microscopies, in vitro cultures (nutrient or Sabouraud agar), and DNA base molecular biology investigations. Specifically, the microbial patina was directly removed by a hydro-alcoholic solution (while evaluating the potential colorimetric change of the artwork’s surface) combined with exposure to EO volatile compounds, performed in a dedicated “clean chamber”. This study proposes, for the first time, the combined use of two plant extracts to counteract microbial development on wooden artworks, providing supplementary information on these products as bio-agents.