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Colorectal cancer is the third most common cancer worldwide. Cancer stem cells are known to play an important role in relapse, and metastases of the disease after chemotherapy. Investigation of new drugs, and their combinations targeting these cells and thus eliminating cancer is one of the most urgent needs of today's chemotherapy. The aim of the present study was to evaluate the effects of Bryophytes like Abietinella abietina (AA), Homolothecium sericeum (HS), Tortella tortuosa (TT), Syntrichia ruralis (SR), and Bryoerythrophyllum rubrum (BR) species extracted with ethyl alcohol on 5-fluorouracil(5-FU) resistant colorectal cancer cell lines (HCT116 and HT29). After extraction, stock solutions of bryophytes were prepared, and IC 50 values were detected in drug-resistant cells obtained with 5-FU application. CD24+, CD44+/CD133+ surface markers and P-glycoprotein (P-gp) mediated efflux were isolated from both 5-FU treated cells and analyzed using the flow cytometry. In all bryophyte-treated groups, the binding Rho123 low (low Rho fluorescence) and Rho high (high Rho fluorescence) were sorted from 5-FU resistant HCT116, and HT-29 cells. All types of bryophytes were found cytotoxic. Bryophyte extract reduced the percentage of Rho low cells in cultures incubated with 5-FU. In summary, the implementation of these bryophytes might be regarded as an effective approach for treatment of colorectal cancer due to their cytotoxic effect that decreases the recurrence of the disease.Supplemental data for this article is available online at https://doi.org/10.1080/01635581.2021.1933098.