Mushrooms have been traditionally used in medicine worldwide among which Laetiporus sulphureus, as an edible mushroom, possess various medicinal properties, such as antioxidative and anticancer. Present study was conducted to evaluate effects of L. sulphureus ethyl-acetate extract (LSEA) on cancer cells viability and concentration of superoxide anion radicals (O₂˙ˉ), nitrites (NO₂ˉ) and reduced glutathione (GSH), as well as its antimigratory potential. Colorectal cancer (HCT-116) and cervical adenocarcinoma (HeLa) cell lines were used and cell viability was assessed by MTT test, while the determination of redox status parameters was done using NBT, Griess and GSH methods. Antimigratory potential was analyzed by Scratch assay 24 h after treatment with two selected sublethal concentrations (10 and 50 μg/mL). For purpose of MTT and redox status analysis, LSEA was applied in 6 different concentrations, for 24 h. Our results showed no significant cytotoxicity of LSEA on HCT-116 and HeLa cells. However, their redox status parameters were affected, whereat LSEA elevated both ROS and RNS in HCT-116 cells, while the GSH level remained almost unchanged. On the other side, HeLa cells were more sensitive to LSEA that was able to trigger production of O₂˙ˉ and NO₂ˉ, especially at higher doses (250 and 500 µg/mL). Regardless, the level of GSH was only slightly heightened by LSEA in this cell line. Moreover, extract attenuated motility of both tested cell lines in both applied concentrations, however its higher concentration (50 µg/mL) had stronger antimigratory effect. LSEA had better effect on motility of HeLa cells by supressing it for more than 50% in higher concentration that also induced significant prooxidative effect in these cells. Due to its promising pre-clinical efficacy, the usefulness of this edible mushroom should be taken into consideration for further studies, esspecially in prevention of cancer.