In this study, the antifungal activity of non-polar to polar extracts from the sea cucumber Holothuria lessoni was evaluated. A total of 45 specimens (mean weight: 275 g) were collected during winter from depths of 5–10 m in the waters surrounding Qeshm Island in the Persian Gulf. Dried sample powder was sequentially extracted using n-hexane (non-polar), diethyl ether (semi-polar), and methanol (polar) solvents. The antifungal activity of the extracts was assessed against Aspergillus fumigatus and Candida albicans by determining minimum inhibitory concentrations (MIC) and minimum fungicidal concentrations (MFC). The n-hexane extract exhibited MIC values of 400 μg/mL against A. fumigatus and 500 μg/mL against C. albicans, with an MFC of 1000 μg/mL for both fungi. The diethyl ether extract showed MFC values of 1000 μg/mL against A. fumigatus and 2000 μg/mL against C. albicans. In contrast, the methanol extract showed no fungicidal activity against either species. Nystatin, used as a positive control, demonstrated MFC values of 50 μg/mL for A. fumigatus and 100 μg/mL for C. albicans.Overall, the results indicate that the non-polar n-hexane extract of H. lessoni possesses higher antifungal activity than the semi-polar and polar extracts. Further studies are warranted to identify the bioactive compounds responsible for this activity and to evaluate their antifungal efficacy in vitro.