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The use of medicinal plants for contraception remains a common practice among South African ethnic groups. The present study assessed the phytochemical profile, cytotoxicity, acute oral toxicity and efficacy of a herbal mixture used for contraception by the Batswana of South Africa. An aqueous extract was prepared from equal quantities (in terms of weight) of Bulbine frutescens (roots), Helichrysum caespititium (leaves) and Teucrium trifidum (leaves) based on a recipe used by traditional health practitioners. The phytochemical profiles of the freeze-dried herbal mixture were analyzed using gas chromatography–mass spectrometry (GC-MS). In addition, cytotoxicity was determined using an MTT assay on Vero cells and in vivo contraceptive efficacy was evaluated using seven Sprague Dawley rats per control and treatment groups. The control group received distilled water while test groups received 5, 50 and 300 mg/kg of the herbal mixture, which was administered orally once a day for three consecutive days. Subsequently, female rats were paired 1:1 with males for 3 days. Their weights were measured weekly and incidence of pregnancy was recorded. The GC-MS chromatogram revealed the presence of 12 identified and 9 unidentified compounds. In terms of safety, the herbal mixture had an IC50 value of 755.2 μg/mL and 2000 mg/kg, which was the highest tested dose that caused no mortality or morbidity in the rats. A contraceptive efficacy of 14.5% was exerted with 50 mg/kg herbal mixture extract while other doses had no effects given that all the rats were pregnant. Based on a chi-square test (p < 0.05), there was no correlation between the tested herbal mixture doses and contraception, nor on the weight of the rats. Overall, the herbal mixture extract was found to be safe but had limited contraceptive efficacy at the tested doses. In future studies, exploring increased dose range, solvent extract types and hormonal analysis will be pertinent.