Because biomarkers (ie, biological markers) can help in the detection and prevention of clinical disease by recognizing changes in biological samples, researchers intended to determine whether there is an association between fluoride concentrations in big toenail (hallux) clippings, hair and the level of fluoride in the water of an endemic and non-endemic fluorosis region. In this case-control study, a total of 48 candidates were involved from an endemic and a non-endemic fluorosis region. According to results, the mean concentration of fluoride in the endemic region of big toenail and hair was significantly higher than the mean concentration of big toenail and hair in the non-endemic region. There is greater observed accuracy in the fluoride assay for big toenails than the fluoride assay for hair. Based on the data from this pilot study, nail and hair samples can be used as biomarkers to detect exposure to biological fluoride. However, hair as a biomarker is less sensitive and specific when comparing the area under the curve values of big toenail and hair samples.