Destruxins (Dtx) are secondary metabolites of the entomopathogenic fungus Metarhizium anisopliae. Recently, Dtx came into focus of interest as anticancer therapeutics. However, data on human and especially on cancer cells are fragmentary. In order to successfully establish novel anticancer therapeutics, a broad knowledge on the cellular and molecular mechanisms underlying their activity is essential. Consequently, this study aimed to investigate the impact of the most common Dtx derivatives A, B and E on human cancer cell growth and survival with a focus on colon cancer cell models. Summarizing, the experimental data showed that (i) Dtx A and B exert potent antiproliferative activity in the micromolar and Dtx E in the nanomolar range in KB-3–1, A549, CaCo-2, and especially in HCT116 colon cancer cells, (ii) all three Dtx derivatives cause imbalance of cell cycle distribution, (iii) their cytostatic/cytotoxic effects are widely p53-independent but reduced by p21- and bax-deletion, respectively, (iv) cytotoxicity is based on intrinsic apoptosis induction and associated with phosphoinositide-3-kinase (PI3K)/Akt pathway inhibition, (v) anticancer activity of Dtx E but not Dtx A and B involves disturbance of the intracellular redox balance, (vi) Dtx inhibit the migration and tube formation of human endothelial cells indicating antiangiogenic potential, and (vii) all three Dtx derivatives possess ionophoric properties not differing in conductivity, ion selectivity and single channel kinetics. Thus, Dtx represent feasible, multifunctional anticancer drug candidates for preclinical development especially against colorectal cancer.