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HIGHLIGHTSThe synthetic fish Epinecidin‐1 (Epi‐1), acts against T. vaginalis both in vitro and in vivo.After one week, the T. vaginalis content was effectively decreased in Epi‐1 treated mice.Epi‐1 is a strong candidate for development as an alternative therapeutic for T. vaginalis infection. ABSTRACT Trichomoniasis is caused by infection with the protozoan parasite Trichomonas vaginalis, and prolonged persistence may lead to serious ill effects in patients. Thus, the development of new therapeutic strategies to combat drug‐resistant T. vaginalis would be clinically beneficial. Antimicrobial peptides (AMPs) comprise an emerging class of molecules that may serve as effective alternatives to antibiotics. In this report, we demonstrate that the synthetic fish AMP, Epinecidin‐1 (Epi‐1), acts against T. vaginalis both in vitro and in vivo. Under in vitro conditions, Epi‐1 disrupted the membrane of metronidazole‐resistant T. vaginalis and completely killed the pathogen. To mimic human infection in vivo, estradiol‐stimulated mice with vaginal Lactobacillus acidophilus colonization were infected with T. vaginalis, followed by treatment with Epi‐1, Vigill, metronidazole or furazolidone. After seven days, the T. vaginalis content was effectively decreased in Epi‐1 treated mice, as measured by acridine orange staining of wet smears and tissue biopsies, as well as qPCR of vaginal discharge DNA. Taken together, our results demonstrate that Epi‐1 is a strong candidate for development as an alternative therapeutic for T. vaginalis infection.