We report the complexation of a potential anticancer agent 2-methoxyestradiol (2-ME) with generation 5 (G5) poly(amidoamine) dendrimers having different surface functional groups for therapeutic applications. The complexation experiment shows that approximately 6–8 drug molecules can be complexed with one dendrimer molecule regardless the type of the dendrimer terminal groups. The bioactivity of 2-ME complexed with dendrimers was found to be significantly dependent on the surface charge of G5 dendrimers. In vitro cell biological assays show that amine, hydroxyl, and acetamide-terminated G5 dendrimers with positive, slightly positive, and close to neutral surface charges, respectively are able to deliver 2-ME to inhibit cancer cell growth. In contrast, 2-ME complexed with carboxyl-terminated G5 dendrimers with negative surface charges does not show its inherent bioactivity. Further molecular dynamics simulation studies show that the compact structure of carboxylated G5 dendrimers complexed with 2-ME does not allow the release of the drug molecules even at a pH of 5.0, which is the typical pH value in lysosome. Our findings indicate that the surface modification of dendrimers with different charges is crucial for the development of formulations of various anticancer drugs for therapeutic applications.