NSC746364, NSC746365, and NSC746366 are structurally novel 2,7-diamidoanthraquinone derivatives compared with other clinically used anticancer agents and have exhibited a unique multilog differential pattern of activity in our earlier studies. To systematically evaluate their potential anticancer activity, three selected compounds were tested for their cytotoxicity in vitro against 60 human cancer lines in the National Cancer Institute's anticancer drug screen as well as for dose response curves and telomerase activity. Cell growth was analyzed by the MTT assay, with differences between dose–response curves analyzed nonparametrically. Telomerase activity was detected by a modified version of the PCR-based assay and telomere repeat amplification protocol assay. To elucidate the structure–activity relationships and in-vitro anticancer activity, we correlated their activity profile [GI50, total growth inhibition (TGI), and LC50] in the screening system and also their effects on telomerase activity, human telomerase reverse transcriptase expression, cell proliferations, and cytotoxicity. As a result we found that NSC746364, NSC746365, and NSC746366 have potent activity with 50% net growth inhibition conferred by 0.23–16.0 μmol/l (2.08 μmol/l mean); 0.78–15.9 μmol/l (2.57 μmol/l mean); 1.38–63.1 μmol/l (3.89 μmol/l mean), respectively. Sensitive cell lines exhibit TGI and 50% lethality to NSC746364, exhibited an LC50 with as little as 2.82 μmol/l and TGI with as little as 0.95 μmol/l; NSC746365, exhibited an LC50 with as little as 3.30 μmol/l, and TGI with as little as 1.65 μmol/l; NSC746366, exhibited an LC50 with as little as 8.80 μmol/l; and TGI with as little as 4.06 μmol/l, respectively. Results of the study extend the initial in-vitro observation reported in the data above and confirm the importance of anticancer activity and telomerase inhibition. The unique molecular characterization, cytotoxicity, and telomerase activity profiles warrant further investigation and indicate a potential novel mechanism of anticancer action involved.