In recent times Gallbladder cancer (GBC) incidences increased many folds in India and are being reported from arsenic hotspots identified in Bihar. The study aims to establish association between arsenic exposure and gallbladder carcinogenesis. In the present study, n = 200 were control volunteers and n = 152 confirmed gallbladder cancer cases. The studied GBC patient’s biological samples-gallbladder tissue, gallbladder stone, bile, blood and hair samples were collected for arsenic estimation. Moreover, n = 512 gallbladder cancer patients blood samples were also evaluated for the presence of arsenic to understand exposure level in the population. A significantly high arsenic concentration (p < 0.05) was detected in the blood samples with maximum concentration 389 µg/L in GBC cases in comparison to control. Similarly, in the gallbladder cancer patients, there was significantly high arsenic concentration observed in gallbladder tissue with highest concentration of 2166 µg/kg, in gallbladder stones 635 µg/kg, in bile samples 483 µg/L and in hair samples 6980 µg/kg respectively. Moreover, the n = 512 gallbladder cancer patient’s blood samples study revealed very significant arsenic concentration in the population of Bihar with maximum arsenic concentration as 746 µg/L. The raised arsenic concentration in the gallbladder cancer patients’ biological samples—gallbladder tissue, gallbladder stone, bile, blood, and hair samples was significantly very high in the arsenic exposed area. The study denotes that the gallbladder disease burden is very high in the arsenic exposed area of Bihar. The findings do provide a strong link between arsenic contamination and increased gallbladder carcinogenesis.