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The antifungal activities of Origanum vulgare essential oil (EO) and of a biocide based on silver and hydrogen peroxide (Sanosil S003) against seven Aspergillus species isolated from different substrata (stone, brick, silk and paper) of cultural heritage objects in Serbia were evaluated. Microdilution, agar dilution and microatmosphere methods were used to determine minimal fungistatic and minimal fungicidal concentrations (MIC and MFC), and light microscopy to determine structural abnormalities. MIC and MFC values for O. vulgare EO ranged from 0.2 to 5 mg mL-1 and for Sanosil S003 from 5 to 250 mg mL-1. Aspergillus sp. sect. fumigati was the most susceptible isolate, where MIC and MFC values were achieved at 0.5 mg mL-1 for O. vulgare EO, while MIC and MFC values for Sanosil S003 were achieved at 5 and 10 mg mL-1, respectively. Morpho-physiological changes were documented in all isolates, including lack of sporulation, depigmentation of conidiogenous apparatus and conidia, and presence of aberrant fungal structures. O. vulgare EO exhibited stronger anti-Aspergillus activity than Sanosil S003, as demonstrated by the higher MIC and MFC values and fewer morpho-physiological changes observed in the tested Sanosil S003 concentrations. O. vulgare EO could be an excellent alternative to commercial biocides, with high potential in the field of cultural heritage conservation.