Objectives: This study was aimed at evaluating the relationship between microscopic agglutination test (MAT) seropositivity and real-time polymerase chain reaction (PCR) reactivity in cattle. An additional objective was assessment of the diagnostic value of both tests in detecting seropositivity and infection caused by pathogenic Leptospira. Methods: Blood and urine samples were collected from 61 bovines in the Los Lagos region of Chile. The MAT detected a panel of eight Leptospira serogroups, whereas real-time PCR was conducted with a TaqMan probe targeting the Leptospira lipL32 gene. Results: The seropositivity rate for pathogenic Leptospira was 39.3% (95% confidence interval [95% CI], 27.04–51.57) according to the MAT, and Tarassovi and Sejroe were the most frequently detected serogroups. The antibody titers ranged from 1:200 to 1:800. The positivity rate determined with real-time PCR was 29.5% (95% CI, 18.05–40.94), and the leptospiral concentrations ranged from 1.07 to 12,500 leptospires per milliliter urine. Among the 24 animals with MAT-reactive serum samples, 18 also had urine samples with positive real-time PCR results; thus 75% of animals had positive results with both techniques. The kappa coefficient between tests of 0.784 (95% CI, 0.539–1.0) indicated substantial agreement. The sensitivity and specificity of real-time PCR versus MAT were 75.0% (95% CI, 57.7–92.3) and 100%, respectively. The positive and negative predictive values were 100% and 86.7% (95% CI, 75.7–96.4), respectively. Conclusion: The results demonstrated the complementary nature of MAT and real-time PCR analyses in diagnosing bovine leptospirosis: combined use of both tests enhanced diagnostic accuracy. Real-time PCR detected Leptospira infection and bacterial renal excretion, thus providing insights into the presence and extent of environmental contamination, whereas the MAT identified seropositivity, antibody titers, and the serogroups associated with infection.