Chronic Kidney disease of uncertain etiology (CKDu) has become a significant disease burden, affecting farming community of Sri Lanka and the exact etiology, which could be multifactorial, is not hitherto established. This study is aimed to determine the association of past hantavirus infection and leptospirosis with the occurrence of CKDu. A cohort (n = 179) of known CKDu patients living in high-CKDu prevalent areas of Anuradhapura district of Sri Lanka was compared with a group of 49 healthy, sex-matched younger blood relatives of CKDu patients (control-1) and another 48 healthy, age, and sex-matched individuals living in low-CKDu prevalent area (control-2) of the same district where same life style and climate conditions prevail. Fifty out of 179 (27.9%) CKDu patients, 16/49 (32.7%) of control-1 and 7/48 (14.6%) of control-2 were found positive for IgG antibodies to Puumala, Hantaan or both strains of hantaviruses. Hantaan strain specificity was found to be predominant in all study groups. Hantavirus IgG sero-prevalence of healthy individuals living in low-CKDu prevalent area was significantly lower compared to CKDu patients and healthy younger blood relatives living in high-CKDu prevalent areas (p = 0.03). Past hantavirus infection possesses a significant risk for the occurrence of CKDu (OR = 4.5; 95% CI-3.1-5.4, p = 0.02). In contrast, IgG seroprevalence to hantaviruses was not significantly different in CKDu patients and healthy younger blood relatives living in high-CKDu prevalent areas indicating past hantavirus infection has no association with the occurrence of CKDu or possibly, younger relatives may develop CKDu in subsequent years. Seroprevalence to leptospirosis showed no significant difference between CKDu patients and healthy controls.