Saccharomyces cerevisiae is a sustainable yeast with many applications in the food industry. Here, we study the use of a Saccharomyces cerevisiae model composed of three different industrial strains (a wine, a beer and a baker’s strain) to assess the antifungal activity of three organic plant-based extracts (Hypericum perforatum 10% w/w, Pistacia lentiscus var. Chia 20% w/w and Rosmarinus officinalis 6% w/w). Three different methods were employed (agar disc diffusion, spot assay, and growth curve analysis). Only the Rosmarinus officinalis extract (6% w/w) exhibited inhibitory activity against all the tested yeast strains in the agar disc diffusion method. In the spot assay, all extracts and their carrier oils (sunflower oil and caprylic triglyceride) exerted similar mild antifungal activity. In the growth curve analysis, all extracts significantly lowered the growth rate of the yeasts, but this was not observed for the carrier oils. The results highlighted that it is important to consider more than one method for testing the antimicrobial activity of different compounds. The three yeast strains exhibited differences in their susceptibility to pharmaceutical antifungals, and the beer and baker’s yeasts were resistant to itraconazole. Moreover, polyphenols were detected in all natural extracts which may be linked to their antifungal activity. Our results suggest that we might consider multiple use of these natural extracts in the food industry as food additives or even preservatives to delay food spoilage.