Class 1 and class 2 fusion peptides bind to the trimeric N-terminal heptad repeat (NHR) and C-terminal heptad repeat (CHR) regions of HIV-1 envelope glycoprotein gp41, respectively, and block its intramolecular folding required for Env-mediated viral and host cell membrane fusion and subsequent viral entry. Using a combination of T-20 (class 1) and (CCIZN17)(3) (class 2), we provide evidence that these classes of fusion peptides work synergistically in an in vitro infectivity assay in inhibiting the entry of primary HIV-1 isolate 89.6 with combination indexes reaching 0.37 and 0.32 at IC(50) and IC(90), respectively. We further demonstrate a similar degree of neutralization synergy between a monoclonal antibody (MAb), D5, targeting the hydrophobic pocket region of the NHR, and 2F5, a well-characterized MAb that targets the C-terminal end of CHR and the membrane-proximal external region (MPER), providing a rational basis for developing combination vaccines targeting these two highly conserved regions of gp41.