Background: Glycyrrhiza (licorice) species are rich in bioactive secondary metabolites and their roots are used traditionally for the treatment of several diseases. In recent years, secondary metabolites of licorice are gaining popularity, especially due to their significant cytotoxic and antitumor effects. However, Glycyrrhiza iconica, an endemic species to Turkey, was not investigated in terms of its anticancer secondary metabolites previously. Purpose: This study aimed to isolate the cytotoxic compounds from G. iconica through bioactivity-guided fractionation and to elucidate mechanisms of action of the most potent compounds. Methods: Total MeOH extract and CHCl 3 , EtOAc, n-buOH and rH 2 O subextracts were prepared from G. iconica roots. Sequential chromatographic techniques were conducted for the isolation studies. The chemical structures of the isolates were established based on NMR and HR-MS analysis. Sulforhodamine B assay was used to evaluate the cytotoxic activity of extracts, main fractions as well as isolates against hepatocellular (Huh7), breast (MCF7) and colorectal (HCT116) cancer cell lines. The mechanisms underlying the cytotoxicity of the most active compounds in Huh7 cells were elucidated by using Hoechst staining, Fluorescence-activated cell sorting and Western blot assays. Results: A new dihydrochalcone, iconichalcone (1) along with 15 known phenolic compounds were isolated from the active CHCl 3 , EtOAc and n-buOH subextracts. Compounds 2–5, 7–16 were found to be responsible for the in vitro cytotoxic activity of G. iconica against all tested cancer cell lines with IC 50 values ranging from 2.4 to 33 µM. Amongst these compounds, licoricidin (10), dehydroglyasperin C (12), iconisoflaven (13) and 1-methoxyficifolinol (15) were found to be the most active compounds according to SRB and real time bioactivity assays and submitted to further mechanistic investigations in Huh7 cells. Compounds 10, 12, 13 and 15 caused accumulation of cells in different phases of cell cycle. Moreover, 10, 12, 13 and 15 induced apoptosis through caspase activation. Besides, 12 showed activation of p53 expression and thus G 2 /M arrest as well as a condensed nuclei, established very promising results. Conclusion: The results demonstrated that the aforementioned compounds, particularly 12 could be potential lead molecules for anticancer drug development that deserve further in vivo and clinical investigations.