The traditions of collecting, processing and applying plant and plant-based medications have been handed down from generation to generation. In many African countries, traditional medicines, with medicinal plants as their most important components, are sold in marketplaces or prescribed by traditional healers (without accurate dose value) in their homes. The value of ethno-medicine and traditional pharmacology is nowadays gaining increasing recognition in modern medicine because the search for new potential medicinal plants is frequently based on an ethno-medicinal basis. The experiment was carried out to isolate and determine the anti-helminthic activity of the molecule (s) isolated from hydro-alcoholic extract of leaves of Azadirachta indica (Neem). The isolation and characterization of the potent molecule was undertaken by chromatographic and spectroscopic techniques. The compound was obtained as a greenish-brown powder after drying and was found to be a tetranor-triterpenoid limonoid. The compound showed Rf value of 0.56 when compared to standard (0.57). The isolated compound showed the retention time of 3.8 minutes very much similar to that of the standard. The compound demonstrated paralysis as well as death of worms in a lesser time at 10 and 20 μg/ml compared to piperazine citrate and albendazole (15 mg/ml). It was observed that anti-helminthic activity was significant as the concentration of the compound varies. The results showed the significant potency of azadirachtin as a significant anti-helminthic.