Congenital cytomegalovirus (cCMV) infection is a major cause of neurodevelopmental impairment and sensorineural hearing loss worldwide, particularly in low- and middle-income countries where standardized molecular screening programs are not routinely implemented. Early laboratory confirmation within the first 21 days of life is essential to differentiate congenital from postnatal infection. This study evaluated the diagnostic agreement between the Simplexa cCMV Direct real-time PCR assay and conventional nested PCR for detecting CMV DNA in neonatal urine specimens in Indonesia. A cross-sectional diagnostic agreement study was conducted between September and December 2023 at a tertiary referral hospital. Urine samples from 87 neonates aged < 21 days with clinical suspicion of congenital infection were tested using both assays. The Simplexa assay detected 29 positive and 58 negative samples, while conventional nested PCR identified 28 positive and 59 negative samples. Agreement was assessed using Cohen’s kappa coefficient, McNemar’s test, and positive and negative agreement rates. The assays demonstrated near-perfect agreement (k = 0.87; 95% CI: 0.75–0.98; p < 0.001). Positive and negative agreement were 91.2% and 95.7%, respectively, with no statistically significant difference between methods (McNemar p = 1.00). Simplexa Direct real-time PCR showed high agreement with conventional nested PCR while offering operational advantages including simplified workflow and reduced contamination risk. These findings support the implementation of automated direct molecular platforms for neonatal cCMV diagnosis in tropical and resource-limited healthcare settings.