Structural organization of actin and actin-binding proteins can be a hallmark of aggressiveness among various populations of solid tumor cells. Here, we studied the impact of chemotherapeutic drugs (paclitaxel and cisplatin) on actin and alpha-actinin structural patterns in human cervical adenocarcinoma cell culture HeLa Kyoto using total internal reflection fluorescence microscopy and single-molecule localization super-resolution microscopy. It was shown that paclitaxel causes disruption of actin cytoskeleton in cancer cells, both for actin and alpha-actinin. We observed disappearance of stress fibers, accumulation of filaments in the cell cortex and morphological changes in focal contacts. With cisplatin, we detected a lower number of thin actin bundles and more dense arrangement of alpha-actinin. Our results suggest that the actin cytoskeleton is a potential target for antitumor chemotherapy.