Juglans neotropica (Peruvian walnut) has been widely used by Andean populations in the form of infusions and decoctions for its antioxidant, hypoglycemic, hepatoprotective, antifungal, and antibacterial properties. This study aimed to investigate the effect of Juglans neotropica extract (JNE) on the spasmolytic activity in rat ileum. Our experiments demonstrated that JNE displayed spasmolytic activity at high concentrations, but did not exhibit antispasmodic effects. Additionally, we determined the juglone concentration in JNE by HPLC-DAD, synthesized juglone using green chemistry and compared its spasmolytic activity with that displayed by JNE. Juglone displayed spasmolytic activity on isolated rat ileal sections. Juglone produced a relaxant effect after contraction by a mechanism involving the inhibition of M2 and M3 muscarinic receptors and the blockade of calcium channels, with no apparent effect on the opening of potassium channels. Finally, in silico molecular docking analyses showed that juglone displayed high docking scores for M2 and M3 muscarinic acetylcholine receptors and CaV1.2 voltage-gated calcium channels. In summary, JNE exhibits a spasmolytic, but not an antispasmodic, effect, whereas juglone demonstrates both spasmolytic and antispasmodic activities. These findings provide a scientific basis for the potential use of juglone for managing intestinal hypercontractility disorders. Further research is needed to elucidate the precise molecular interactions to explore its therapeutic applications in gastrointestinal dysfunction.