Purpose: The present study aimed to investigate the effects of α-Klotho and oxidative stress markers on urinary stone disease (USD) and demonstrate their use as biochemical markers in USD. Methods: Among the 90 individuals included, 30 individuals were healthy controls (Group 1), 30 individuals presented with USD for the first time (Group 2), and 30 individuals demonstrated recurrent USD (Group 3). Serum levels of α-Klotho, vitamin D, malondialdehyde (MDA), total oxidant status, and total antioxidant status were determined using spectrophotometry analysis. Serum calcium and parathormone levels and 24-h urine calcium levels were measured via biochemical analysis. Results: No significant intergroup difference was noted in terms of age and sex. The groups had significant differences regarding α-Klotho, oxidative stress index (OSI), MDA, and 24-h urine calcium levels. α-Klotho was a determinant of 24-h urine calcium level and OSI. An increase of 1 pg/mL in α-Klotho level appeared to result in a decrease of 8.55 mg in 24-h urine calcium level and a decrease of 0.04 Arbitrary Unit in OSI. In patients experiencing USD for the first time, α-Klotho values were < 21.83 pg/mL and showed 66% sensitivity and 64% specificity. In individuals with recurrent stone formation, α-Klotho values below 19.41 pg/mL had 60% sensitivity and 77% specificity. Conclusions: The biochemical markers investigated herein, i.e., α-Klotho, OSI, and MDA, were involved in the pathogenesis of stone formation and can be used in day-to-day clinical practices of urology clinics to identify patients at risk for both first time and recurrent USD.