High consumption of red chili pepper has been shown to be a risk factor for gallbladder cancer (GBC) inChilean women with gallstones, and included mutagens may be important in this context. We aimed to investigatethe mutagenicity and mutagens in Chilean red chili pepper in the Ames test using Salmonella typhimurium strainsTA98, TA1537, TA100, and TA1535 with and without metabolic activation (S9 mix). Pure capsaicin was testedfor mutagenicity using strain TA98. The presence of aflatoxins was evaluated by two-dimensional thin layerchromatography, and then the concentrations of aflatoxins B1, B2, G1, and G2 were measured by an HPLCsystem. In strain TA98, the mean numbers of revertant colonies with and without the S9 mix were 2.5- and 2.2-fold higher than those of each negative control, respectively. However, pure capsaicin did not show mutagenicactivity in strain TA98. Aflatoxin contamination of red chili pepper was confirmed, and the concentrations ofaflatoxins B1 and G1 were 4.4 ng/g and 0.5 ng/g, respectively. Our findings suggest that low-level but protractedexposure to aflatoxins may be associated with the development of GBC in Chilean women who carry gallstones.