Naphthalimide-benzimidazole conjugates were prepared using two different types of spacer units; either a simple alkane chain or a substituted piperazine moiety with variable alkyl side chains. Each set of conjugates was evaluated for their in vitro anticancer activity, and compounds 14a, 14b and 20c were found to exhibit significant activity against a number of cancer cell lines. In particular, compound 14a showed remarkable anticancer activity with GI50 values of 0.02 μM and 0.49 μM against central nervous system (SNB-75) and leukemia (K-562) cell lines, respectively. Compound 14b showed noticeable activity against melanoma (MAME-3M) cell line with GI50 value 0.09 μM. Compound 20c also displayed selectivity against leukemia cell lines with GI50 values up to 0.21 μM as well as appreciable broad-spectrum cytotoxicity.