Retrocyclins are cyclic antimicrobial peptides that exhibit potent activity towards a broad range of primary and laboratory-adapted strains of human immunodeficiency virus type 1 (HIV-1) in vitro. The current study shows that RC-101, an analogue of retrocyclin, prevented HIV-1 infection in an organ-like construct of human cervicovaginal tissue and retained full activity in the presence of vaginal fluid. The peptide remained within the cervicovaginal tissues throughout the 9-day incubation period without altering tissue viability, inducing damage or inducing the release of inflammatory cytokines. Collectively, these data support the potential development of RC-101 as a topical microbicide to prevent HIV-1 infection and transmission.