A series of neomycin dimers have been synthesized using click chemistry with varying functionality and length in the linker region to target the human immunodeficiency virus type 1 (HIV-1) TAR RNA region of the HIV virus. The TAR (Trans-Activation Responsive) RNA region, a 59 bp stem-loop structure located at the 5'-end of all nascent viral transcripts, interacts with its target, a key regulatory protein, Tat, and necessitates the replication of HIV-1. Neomycin, an aminosugar, has been shown to exhibit multiple binding sites on TAR RNA. This observation prompted us to design and synthesize a library of triazole-linked neomycin dimers using click chemistry. The binding between neomycin dimers and TAR RNA was characterized using spectroscopic techniques, including FID (fluorescent intercalator displacement), a FRET (fluorescence resonance energy transfer) competitive assay, circular dichroism (CD), and UV thermal denaturation. UV thermal denaturation studies demonstrate that binding of neomycin dimers increases the melting temperature (T(m)) of the HIV TAR RNA up to 10 °C. Ethidium bromide displacement (FID) and a FRET competition assay revealed nanomolar binding affinity between neomycin dimers and HIV TAR RNA, while in case of neomycin, only weak binding was detected. More importantly, most of the dimers exhibited lower IC(50) values toward HIV TAR RNA, when compared to the fluorescent Tat peptide, and show increased selectivity over mutant TAR RNA. Cytopathic effects investigated using MT-2 cells indicate a number of the dimers with high affinity toward TAR show promising anti-HIV activity.