Objective: The limitations encountered in the management of fungal infections are due to the resistance, high toxicity, and overuse of conventional antifungal drugs. For bringing solutions, the antifungal activity of Aeollanthus heliotropioides essential oil will be evaluated and optimized. Material and methods: The aerial parts of A. heliotropioides were harvested and essential oil extracted by hydrodistillation. The chemical composition was determined using gas chromatography and gas chromatography coupled with mass spectrometry and nuclear magnetic resonance. The sensitivity of fungal strains was determined using broth microdilution method. The fungicidal parameters were checked by viability assay using methylene blue dye. The Fractional Inhibitory Concentration Index was determined according the two-dimensional checkboard methods. The efficiency of the simulated optimum concentrations confirmed experimentally on American type culture collection strains, through the Time Kill Kinetic Study. Results: The yield of extraction of essential oil was 0.1%. The major compounds were linalool (38.5%), Z-α-farnesene (25.1%), 9-hexa-decen-1-ol (13.9%) saturated/unsaturated massoia and γ-lactones (4.5%). The MIC of extract on yeast isolates ranged from 0.6mg/mL to 5mg/mL. The combination of essential oil with thymol leads mainly to synergistic effects (0.5≤FICI). The optimums of essential oil (1.6±0.4μl/mL) and thymol (0.6±0.1mg/mL) revealed a total inhibition of yeast after 120 and 180minutes according to the yeasts strains used. Conclusion: This study highlights the in vitro antifungal activity of A. heliotropioides essential oil and it synergistic effect with thymol.