Gallbladder cancer (GBC) is an aggressive malignancy, with gallstone disease (GSD) recognized as the primary risk factor. Although the precise mechanism linking GSD to GBC remains unclear, evidence suggests that gallstone characteristics play a significant role. This study investigates the physicochemical characteristics of gallstones critical for GBC development. We analyzed 40 gallstone samples from 30 GSD and 10 GBC with GSD (GBCGS) patients using advanced spectroscopic and imaging techniques such as fourier transform infrared (FTIR), powder X-ray diffraction (PXRD), nuclear magnetic resonance (NMR), and scanning electron microscopy energy-dispersive X-ray (SEM-EDX)). Subsequently, elemental analysis of 10 gallstones each from GBCGS and GSD was conducted via inductively coupled plasma-mass spectrometry (ICP-MS). Gallstones from the GSD group were identified as cholesterol (70%), mixed (13.3%), pigment (6.7%), and calcium carbonate (10%), while the GBCGS group included only cholesterol (70%) and mixed (30%) types. Cholesterol was the dominant organic component in most gallstones, with the cholesterol and mixed types exhibiting highly crystalline phases characterized by a stacked plate-like microstructure, particularly prominent in the GBCGS group. Additionally, the GBCGS group revealed significantly higher concentrations of carcinogenic elements such as arsenic, chromium, mercury, iron, and lead (p < 0.05), suggesting their accumulation in the gallbladder and gallstones. Consequently, our findings highlight that the physicochemical properties of cholesterol-rich gallstones and exposure to carcinogenic elements play a key role in the pathogenesis of GBC in Assam. These results emphasize the need for further research into cholesterol dysregulation and its link to elemental toxicity.