Herbal medicines are widely utilized in Ghana for the management of malaria, with polyherbal formulations increasingly integrated into mainstream healthcare. However, most of these products are prescribed without data on quality, safety and efficacy, and this has raised concerns among the scientific community. Obiri Mixture, a registered polyherbal product, and used for the management of uncomplicated malaria. The antimalarial activity of Obiri Mixture was evaluated in vitro against chloroquine-sensitive (3D7) and resistant (Dd2) Plasmodium falciparum strains using the SYBR Green I assay. Cytotoxicity was assessed on human erythrocytes using the MTT assay. Acute oral toxicity was assessed in rats at 2000 mg/kg following OECD guidelines. In vivo antimalarial efficacy was evaluated using the 4-day suppressive and Rane’s curative tests in Plasmodium berghei-infected mice. Phytochemical, physicochemical, elemental, and microbial analyses were performed according to standard procedures. Obiri Mixture exhibited potent in vitro activity with IC50 values of 1.196 µg/mL (3D7) and 0.325 µg/mL (Dd2), and a CC50 >100 µg/mL, with a high selectivity index. No mortality or observable toxicity was recorded at 2000 mg/kg in the acute toxicity study. There was significant (p < 0.05) suppression of parasitaemia, achieving >78% suppression and >82% in the curative assay at 300 mg/kg, with prolonged survival of treated mice. Phytochemical screening revealed secondary metabolites. Physicochemical parameters, elemental content, and microbial load were within WHO-recommended limits. Obiri Mixture demonstrates potent antimalarial activity, safety profile, and acceptable quality standards. This provides scientific support for its use and potential integration into evidence-based malaria management.