Background: This article describes the analysis and interpretation of data relating to the presence of cadmium, lead, mercury and fluoride in human bone samples obtained from cadavers of patients dying of Chronic Kidney Disease of uncertain aetiology (CKDu) in a case-control study, which the authors believe to be the first in Sri Lanka. Methods: This is a case-control study comparing selected nephrotoxins levels in autopsy samples of bones from persons confirmed to have died of CKDu, and who had lived in a CKDu hotspot and controls with no history of abnormal kidney functions who had lived in areas having a low prevalence of CKDu. Results: The average age at death of the cases was 59.6 ± 15.1 (±SD), while that for controls was 58.0 ± 19.3 (±SD) years with no significant statistical difference. Calcium adjusted bone lead and bone fluoride levels were significantly higher among CKDu cases (n = 14) than those of controls (n = 33). Further, younger and older clusters of CKDu cases can be differentiated when the calcium adjusted lead content was considered. Younger patients reported higher lead content compared to the older group. Cadmium and mercury content did not show a remarkable difference among cases and controls. Conclusions: Our results show a significant accumulation of lead in bone tissue in persons who have died of CKDu which indicates a higher chronic exposure of the CKDu victims, to lead. Fluoride content in the bones of cases is also significantly high. The results indicate that a gradual reduction of glomerular filtration rate (GFR) may have occurred with the chronic exposure to lead. At a certain point of the threshold of low GFR, excretion of fluoride may be impaired, since the main excretory pathway of fluoride is via the kidneys. Fluoride accumulation in blood in turn may exacerbate nephrotoxicity, triggering a synergistic cascade of events which may lead to a further deterioration in the GFR. The extremely high fluoride content detected suggests a cumulative effect in people exposed to comparatively higher fluoride levels, most probably from drinking water.