BackgroundThe unmet demand for effective malaria transmission-blocking agents targeting the transmissible stages ofPlasmodiumnecessitates intensive discovery efforts. In this study, a bioactive bisbenzylisoquinoline (BBIQ), isoliensinine, fromCissampelos pariera(Menispermaceae) rhizomes was identified and characterized for its anti-malarial activity.MethodsMalaria SYBR Green I fluorescence assay was performed to evaluate the in vitro antimalarial activity against D6, Dd2, and F32-ART5 clones, and immediate ex vivo (IEV) susceptibility for 10 freshly collectedP. falciparumisolates. To determine the speed- and stage-of-action of isoliensinine, an IC50speed assay and morphological analyses were performed using synchronized Dd2 asexuals. Gametocytocidal activity against two culture-adapted gametocyte-producing clinical isolates was determined using microscopy readouts, with possible molecular targets and their binding affinities deduced in silico.ResultsIsoliensinine displayed a potent in vitro gametocytocidal activity at mean IC50gamvalues ranging between 0.41 and 0.69 µM forPlasmodium falciparumclinical isolates. The BBIQ compound also inhibited asexual replication at mean IC50Asexualof 2.17 µM, 2.22 µM, and 2.39 µM for D6, Dd2 and F32-ART5 respectively, targeting the late-trophozoite to schizont transition. Further characterization demonstrated a considerable immediate ex vivo potency against human clinical isolates at a geometric mean IC50IEV= 1.433 µM (95% CI 0.917–2.242). In silico analyses postulated a probable anti-malarial mechanism of action by high binding affinities for four mitotic division protein kinases; Pfnek1, Pfmap2, Pfclk1, and Pfclk4. Additionally, isoliensinine was predicted to possess an optimal pharmacokinetics profile and drug-likeness properties.ConclusionThese findings highlight considerable grounds for further exploration of isoliensinine as an amenable scaffold for malaria transmission-blocking chemistry and target validation.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12936-023-04590-7.