Streptococcus pneumoniae is a commensal of the human upper respiratory tract. In certain cases, it can lead to serious invasive infections peaking in very young children and the elderly. Especially young children are frequent carriers and are thus regarded as the reservoir for horizontal transmission of pneumococci. This is the first study evaluating pneumococcal colonization patterns in healthcare professionals working in a tertiary care pediatric hospital, including carriage prevalence, serotype distribution, and risk factors for carriage. One oropharyngeal and one nasal swab per individual were directly plated onto appropriate agar plates and conventional culture was used for bacterial identification. Pneumococcal isolates underwent serotyping using Neufeld’s Quellung reaction with type-specific antisera. Additional nasal and oropharyngeal swabs were taken for qPCR analysis targeting lytA. In total, 437 individuals were enrolled. S. pneumoniae was isolated in 4.8% (21/437) of the study cohort using conventional culture and in 20.1% (88/437) of subjects using qPCR. Independent risk factors for pneumococcal carriage were living in the same household with children under 8 years of age and being aged 36–45 years with a carriage prevalence reaching 11.6% (vs. 2.9%, p = 0.002) and 6.7% (vs. 4.3%, p = 0.029), respectively. The most common serotypes were 6C and 3. A total of 71.4% (15/21) of the detected serotypes are not included in any currently available pneumococcal vaccine; 28.6% (6/21) of the carried serotypes are included in the PCV13 vaccine. We found a relevant amount of pneumococcal carriage bearing the potential risk of horizontal in-hospital transmission.