To improve the anticancer activity of commercial drugs, a series of well-defined Janus dendrimer conjugates with chlorambucil (CLB) and naproxen, which feature diethanolamine groups and ethylene glycol in the structure, were synthesized and characterized in detail. The anticancer activity of all synthesized dendrons and dendrimers were tested and screened against six human cancer cells. In vitro all conjugates at 25 μM exhibit selectivity towards human mammary adenocarcinoma (MFC-7) cells as compared to U-251, PC-3, and HCT-15 cells, while being only slightly active in SKLU-1 and K-562 cells. The IC50 values showed that the best growth suppressor of MFC-7 (4 ± 0.3 μM) was the second-generation dendrimer with four chlorambucil and four naproxen moieties. The result indicates that Janus dendrimers are promising molecular scaffolds for the design of more potent anticancer drugs. Further biological evaluations, such as the anticancer activity of the Janus dendrimers in vivo, are ongoing.