Objective: The aim of this research was to examine the potential cytotoxic effect of Cantharellus cibarius mushroom aqueous extracts obtained at 25°C and 70°C on the ovarian cancer cell line OVCAR-3. It was assumed that the number of ovarian cancer cells, after treatment with aqueous extracts of mushroom, will decrease compared to the control group. Materials and methods: Cytotoxic activity was examined using the MTT assay. The extracts were prepared in 6 different concentrations: 1 µg/mL, 50 µg/mL, 100 µg/mL, 250 µg/mL, 500 µg/mL and 1000 µg/mL, and the effect was observed after 24, 48 and 72 hours of incubation. The effectiveness of Cantharellus cibarius mushroom extracts was determined spectrophotometrically at 570 nm. The percentage of metabolically active cells was determined by comparing the absorbance of the formed formazan in cells treated with Cantharellus cibarius mushroom extracts (test group) and untreated cells (control group). Results: The results are graphically presented in relation to cell viability (%) and extract concentration. Aqueous extracts of Cantharellus cibarius mushroom obtained at 25°C and 70°C showed similar effects. For both extracts, the most significant results were achieved after 72 hours of incubation at concentrations of 500 µg/mL and 1000 µg/mL. The extract obtained at 25°C at a concentration of 500 µg/mL reduced the cell viability to 81.5%; at a concentration of 1000 µg/mL, the cell viability was 68%. The extract obtained at 70°C at both concentrations reduced cell viability to 69%. Conclusion: Aqueous extracts of Cantharellus cibarius mushroom obtained at 25°C and 70°C led to a decrease in cell viability of in vitro exposed ovarian cancer cells of the OVCAR-3 cell line. The examined extracts mainly show cytotoxic activity depending on the exposure time and the concentration of the extract. In some cases, the cytotoxic activity of the mushroom extracts is not proportional to the increase in the concentration of the extract, and the exposure time and in some cases, there was a slight increase in the number of metabolically active cells. Aqueous extracts of Cantharellus cibarius mushroom obtained at 25°C and 70°C show a similar cytotoxic effect on ovarian cancer cells of the OVCAR-3 cell line. Both extracts achieve significant results after 72 hours of incubation at concentrations of 500 µg/mL and 1000 µg/mL. Mushroom extracts did not reach the IC50 value. The obtained results show the cytotoxic effect of Cantharellus cibarius mushroom aqueous extracts, but additional in vitro and in vivo studies are needed to confirm the obtained results and cytotoxic activity.