This work analyzed the influence of different application protocols on the efficacy of two biocides against the foliose lichen Xanthoparmelia tinctina on the sandstones of the Roman Archaeological site of Luni (Italy). The hypotheses that (a) biocide application tools (brush vs. poultice), (b) pre-treatment hydration, and (c) post-treatment washing may affect devitalization success were verified by monitoring chlorophyll a fluorescence of thalli, both in situ and in laboratory conditions. The hypothesis that (d) stone substrate may act as reservoir for later biocide release under repeated cycles of wetting and drying was also assayed. Analyses confirmed the importance of the application tool, with cellulose poultice being more effective than brush. Hydration influenced the biocide absorption by thalli. Moreover it modulated the metabolic activity and susceptibility to the available toxic compound, hindering lichens from entering a dormant state to tolerate stress. Depending on the preparation solvent (water vs. white spirit), the biocide application benefited from pre-treatment hydration and/or a post-treatment washing. Lastly, we showed that different sandstones variously adsorb the biocides and potentially contribute as a reservoir for their long-term release at low concentrations during successive hydration events. Display omitted •The protocol to apply biocides on lichens affects the devitalization effects.•Cellulose poultice application of biocides is more effective than that by brush.•Pre-hydration and/or post-washing of thalli regulate biocide effectiveness.•The stone substrate acts as a reservoir for long-term release of biocide.•Effective application protocols can limit useless chemical release to the environment.