Intrauterine Ureaplasma infection is associated with chorioamnionitis and preterm birth. The difficulty of detecting Ureaplasma species by conventional culture methods makes definitive diagnosis of clinical infection challenging. Thus far, quantitative tests for Ureaplasma have been performed using adult cervical samples, amniotic fluid, and pediatric bronchial secretions, but quantification of bacterial count in central nervous system infections caused by Ureaplasma species has not been unreported. We report a case of culture-negative Ureaplasma meningitis in a preterm infant in whom novel techniques to identify this pathogen and quantify bacterial count were effective. We suspected meningitis based on a sustained reduction in cerebrospinal fluid (CSF) glucose levels. Multiple CSF cultures were sterile. We confirmed infection by Ureaplasma species using the melting temperature mapping method. Treatment with erythromycin and ciprofloxacin resulted in a gradual decrease in the bacterial count in the CSF to 0. Our study highlights the potential utility of the melting temperature mapping method as a new diagnostic tool for culture-negative Ureaplasma meningitis and establishes the utility of serial quantification of bacterial count to monitor response to therapy.