Knowledge of the chemical, structural, and elemental composition of gallstones is essential for etiopathogenesis of gallstone disease. To identify the predisposing factors for gallstone formation, X-ray diffraction powder analysis, atomic absorption spectroscopy, and various biochemical estimations were carried out. In the present study, trace elemental analysis revealed calcium as the major constituent element in addition to the iron, magnesium, and zinc in the majority of the gallstones. Patients with gallstones exhibited increased serum total bilirubin and conjugated bilirubin levels and liver function parameters (serum glutamic pyruvic transaminase, serum glutamic oxaloacetic transaminase, and alkaline phosphatase). In patients with gallstones, higher concentrations of malondialdehyde, significantly higher glutathione disulfide/glutathione (GSH) ratio, reduced total GSH levels, and significantly decreased antioxidant enzymes activities (superoxide dismutase, catalase, and glutathione peroxidase) were found than in patients without gallstones. Further studies are needed to establish whether the observed differences are a cause or an effect of gallstone formation. Such studies could ultimately result in the development of new strategies for the treatment of gallstones and might provide clues for prevention of gallstones formation.