Impaired gallbladder motility is a contributing factor to gallstone formation. Since many drugs delaying intestinal motility inhibit gallbladder emptying, the aim of the present study was to evaluate the effect on gallbladder and sphincter of Oddi motility of a Natural Chestnut Wood Extract (NEC) that reduces intestinal motility. In order to evaluate the effect of the extract in normal- and high-risk gallstone conditions, the investigation was performed using tissues from animals fed normal and lithogenic diet. Fifty guinea pigs were administered either control or lithogenic diet. The spontaneous motility of the gallbladder and sphincter of Oddi were recorded on isolated gallbladder tissues; thereafter, the effect of NEC on motility was tested and compared with carbachol (CCh), potassium chloride (KCl), noradrenaline (NA), and A71623. Compared to controls, the lithogenic diet induced an irregular and disordered motor pattern in both the gallbladder and sphincter of Oddi. NEC increased gallbladder and decreased sphincter of Oddi spontaneous motility independently of cholinergic, adrenergic, and CCK-1 receptor-mediated pathways both in controls and in lithogenic diet-fed animals, although the effect was lower in the latter group. The effect was reversible and mediated by calcium channels. The natural extract of chestnut increasing gallbladder contraction and inducing the relaxation of the sphincter of Oddi can be of benefit in pathological conditions associated with increased transit time at risk of gallstones.