<p>The activity of six essential oils was investigated against eight fungal isolates (four <i>Aspergillus</i> and four <i>Penicillium</i> species) isolated from cultural heritage conservation premises in Serbia. To analyze the chemical composition of essential oils (EOs), gas chromatography coupled with mass spectrometry was employed. The antifungal activity of selected EOs was investigated using microdilution and microatmosphere methods while the commercial biocide benzalkonium chloride (BAC) was used as a control. Furthermore, molecular docking was used as an efficacious in silico method for the determination of interaction between dominant EO compounds and enzyme CYP51, essential for fungal ergosterol synthesis. It was demonstrated that BAC, <i>Cinnamomum zeylanicum</i>, and <i>Gaultheria procumbens</i> EOs had the strongest antifungal activity, which is in concordance with the results of molecular docking. Namely, the highest energy of enzyme–cofactor interaction was obtained for eugenol (the dominant component of <i>Syzygium aromaticum</i> and <i>C. zeylanicum</i> EOs). Moreover, it was found that the most resistant fungal isolates were <i>A. flavus</i> and <i>A. niger</i>, while <i>A. sydowii</i> and <i>P. citrinum</i> were the most susceptible. The results of our study point to the possibility of using studied environmentally friendly biocides of biological origin for the preservation of historical monuments and artifacts.</p>