Colorectal cancer (CRC) is one of the leading causes of cancer-related death worldwide. The incidence depends on geographical location, lifestyle, and genetic factors. CRC is manifested through the formation of adenomatous polyps on the inner wall of the colon. CRC is treated with surgery, radiation, chemotherapy, or a combination of several options. 5-FU is one of the leading chemotherapeutic options for the treatment of CRC, but due to the frequent appearance of drug resistance in tumor cells, it is necessary to consider new treatment options. Phytochemicals show promising potential thanks to their wide spectrum of positive effects, among which is antitumor activity. The aim of this work was to investigate the antitumor activity of the phytochemicals rutin and quercetin, and their effect in combination with 5-FU in HCT116 colon tumor cells. 24-hour treatments of rutin, quercetin, 5-FU and their combinations were used, the effects of which were tested using XTT, Western blot and immunofluorescence. The results showed the cytotoxic effect of rutin and quercetin, which was enhanced in combination with 5-FU, the activation of autophagy and the intrinsic and extrinsic pathway of apoptosis, with increased activation in co-treatments with 5-FU, and the impact on the ERK/MAPK signaling cascade and the FoxO3a transcription factor. It has been shown that combination therapies are more effective than separate therapies, and that rutin combined with 5-FU is the most effective combination. Rutin, although of a more modest effect in a separate treatment, acts as a chemosensitizer of 5-FU, enhancing its effect on tumor cells. However, it is necessary to carry out research in vivo to establish the action of phytochemicals separately and in combination with chemotherapeutics inside the organism in a more detailed manner.