Unwanted pregnancies and sexually transmitted infections (STIs) are major health concerns of women worldwide. These concerns have prompted efforts to develop Multipurpose Prevention Technologies (MPTs), which simultaneously provide contraception and prevent STIs, including HIV. LL-37, the only human cathelicidin and an effective spermicide on human sperm, has broad antimicrobial activity including in vitro activity against HIV. 17BIPHE2 is a truncated LL-37 peptide, engineered to contain 5 unnatural residues, thus limiting its protease degradation within vaginal fluid. Hence, this antimicrobial peptide (AMP) represents a promising MPT agent. It was therefore hypothesized that these peptides would be inhibitors of HIV infection in cell lines, PBMC, and CD4+ T cells. In the chronically infected ACH-2 cell line, there was significant reduction in p24 production when cells were treated with 17BIPHE2, but not LL-37. When 17BIPHE2 was pre-incubated with HIV prior to infection and present during infection, viral replication decreased in the TZM-bl reporter cell line, but this result was not recapitulated in the primary activated cells, PBMCs nor isolated CD4+ T cells. Conversely, pre-incubation of 17BIPHE2 with target cells prior to infection significantly inhibited HIV infection in a dose-dependent manner. Therefore, 17BIPHE2 may act on the cell or on the virus/cell interaction rather than on the virus itself to inhibit HIV infection.