Neomycin is known to preferentially bind to A-form nucleic acid structures including triplex DNA, DNA and RNA hybrid, and duplex RNA. Tethering a DNA intercalator moiety to the C5” position of the ring III of neomycin is a practical approach to develop potent binders targeting various nucleic acid secondary structures via a synergistic effect; however, the minimal stacking surface of the intercalating moiety required to exhibit the effect remains unclear. In the present work, we synthesized four nucleobase and neomycin conjugates via click chemistry. All four conjugates stabilized a DNA oligonucleotide triplex in the thermal denaturation experiments monitored by UV. The guanine-neomycin conjugate (6b) showed a better triplex stabilization effect than neomycin. All the conjugates, as well as neomycin, exhibited no thermal stabilization effect on a human telomeric DNA G-quadruplex. These results suggest that the synergistic effect of binding is vastly dependent on the surface area of the stacking moiety of the conjugates. In addition, tethering a nucleobase to the C5” position of neomycin enhanced the cytotoxicity of neomycin toward MCF-7 and HeLa cancer cells but decreased the antibacterial effect of neomycin against several Gram-negative and Gram-positive bacterial species.