A series of carbazole–pyrrolobenzodiazepine conjugates (4a–g and 5a–f) have been designed, and synthesized as anticancer agents. These compounds are prepared by linking the C8-position of DC-81 with a carbazole moiety through simple alkane spacers as well as piperazine side-armed alkane spacers in good yields. The DNA binding ability of these conjugates has been determined by thermal denaturation studies and also supported by molecular docking studies. These conjugates showed potent anticancer activity with GI50 ranging from 5.27–0.01 mM. The FACS analysis and BrdU assay of selected conjugates (4c, 4f, 5a and 5f) on MCF-7 cell lines disclosed the increased G1 cell cycle arrest and one of the conjugates 5f has exhibited significant anticancer activity. The analysis of the intrinsic factors involved in causing the G1 arrest in MCF-7 cell lines by 5f conjugate has been demonstrated on the proteins which play a vital role in G1 arrest followed by apoptosis (Cyclin D1, CDK4, c-Jun, JunB, CREB, p53, JNK1/2, procaspase-7, cleaved PARP, pRb, and BAX). Thus, these PBD conjugates (in particular 5f) have promising potency for combating human carcinoma.