Gallbladder cancer (GBC) is one of the deadliest malignancy and treatment options are deplorably limited. Better strategies of prevention are urgently needed but knowledge on risk factors remains scarce. Recent data suggested that arsenic (As) may be involved in GBC carcinogenesis but the question remains debated. To date, there are no data on As measurement in GBC samples. This pilot study aimed to measure As concentrations in tissue samples from patients with GBC compared to non-cancerous gallbladder (NCGB). Included patients underwent cholecystectomy at Hospital Clinico Universidad de Chile, Santiago in Chile, a country with high As exposure, between 2001 and 2020. Tissue samples were preserved in formalin-fixed, paraffin-embedded blocks. Selected samples were retrieved, processed and submitted to inductively coupled plasma mass spectrometry (ICP-MS) to determine As concentrations. A total of 77 patients were included, including 35 GBC and 42 NCGB. The two groups were comparable, except for age (68 vs. 49 years, p < 0.001). Measured in 11 GBC and 38 NCGB, total As was detected in 5 GBC (14%) compared to 0 NCGB samples (p < 0.001). GBC group also showed higher median values of As compared to NCGB (p < 0.001). This pilot study provided a proof-of-concept to measure As concentrations in gallbladder samples and showed higher level of As in GBC samples compared to NCGB, paving the way for future studies aiming to investigate the impact of As on GBC, which may contribute to the prevention of this deadly disease.