In this study, the biocidal activity of two plant derivatives (oregano and cloves’ essential oils—EOs) was evaluated, as a potential innovative and eco-friendly cleaning method for canvas paintings. The object of the study was the oil painting on canvas entitled “Studio di nudo” (Giovanni Maria Mossa, 1921), showing stains caused by microorganisms. The research focused on: (1) isolation and identification of microorganisms associated with discolorations on the obverse and reverse sides of the canvas; (2) evaluation of biocidal activity of selected EOs against fungal and bacterial collections. The phylogenetic identification was conducted with both cultivation and molecular methods. The canvas was mainly colonized by Penicillium, Aspergillus, and Cephaloteca fungal genera and by bacteria of the Bacillus genus. To evaluate the biocidal effect of the EOs’ volatile components only, an antibiogram assay (agar disc diffusion method) and a customized assay (named the contactless test) were conducted. Tested EOs showed antimicrobial activity on fungi and bacteria. However, compared to cloves, oregano EO exhibited a better inhibition activity both in contact and contactless tests. The work is pioneering for the use of EOs’ volatile compounds against oil painting biodeteriogens, and gives insights into possible extended, innovative and eco-friendly cleaning methods for painting control procedures.