The development of a simple co-culture system provides a more effective examination of the therapy impact on tumor cells in the presence of desired microorganisms. Laetiporus sulphureus is an edible mushroom, used worldwide with known medicinal potential. The effect of ethyl acetate (EA) extract of L. sulphureus was evaluated on the viability of cervical adenocarcinoma cells (HeLa) and migratory potential of S. boulardii in a co-culture system. Then, we examined the effect of Saccharomyces boulardii on the viability of these cells in a co-culture system. Co-culture system was formed in a test tube of 50 mL consisted of 40 mL of agar with yeast, and HeLa cells on the coverslips. Cells were seeded on the top side of the coverslips then coverslips were placed upside down towards the yeast agar. 10 mL of Dulbecco's modified Eagle's minimal essential medium (DMEM) (Control), or 10 mL of EA at a concentration of 10 µg/mL were added for control or treatment. Cell viability was determined with trypan blue staining after 12 and 24 h. Yeast migration was measured using the ImageJ program. Our results showed no cytotoxicity of EA extract on HeLa cells. However, under the influence of probiotic species S. boulardii, the viability of these cells was significantly reduced. Extract of L. sulphureus did not significantly reduce cell viability in comparison to untreated co-culture system. These results indicate a potential cytotoxic effect of probiotic species on cervical adenocarcinoma cells. This effect could result from the metabolic activity of S. boulardii cells which seems to be further enhanced following the physical contact between yeast and cancer cells, after migration of S. boulardii through agar toward cancer cells. Also, extract of L. sulphureus induced strong yeast migration in coculture after 12 h. Further studies should be conducted regarding this mushroom in a co-culture system with S. boulardii.