A series of new podophyllotoxin linked β-carboline congeners have been synthesized by coupling various substituted β-carboline acids with 4β-aminopodophyllotoxin. Evaluation of their anticancer activity against a panel of human cancer cell lines such as lung cancer (A549), prostate cancer (DU-145), MDA MB-231 (breast cancer), HT-29 (colon cancer) and HeLa (cervical cancer) suggested that 7i and 7j are the most cytotoxic compounds with IC50 values of 1.07 ± 0.07 μM and 1.14 ± 0.16 respectively against DU-145 cell line. Further, detailed biological studies such as cell cycle analysis, topoisomerase II inhibition, Comet assay, DNA binding studies and docking studies have revealed that these congeners are DNA interacting topoisomerase II inhibitors.