OBJECTIVE: We investigated the presence of mycobacteria in the oral cavity and oropharynx of children with nontuberculous mycobacterial (NTM) cervicofacial lymphadenitis compared to negative controls. Our aim was to evaluate whether oral cavity and oropharyngeal swab sampling could serve as a minimally invasive alternative to diagnostic sampling of excised lymph node tissue or punctate. METHODS: Children who underwent surgery under general anesthesia due to a strong clinical suspicion of NTM cervicofacial lymphadenitis were enrolled in this multicenter cross-sectional study. The control group comprised children undergoing surgery under general anesthesia for reasons other than lymphadenopathy. The obtained oropharyngeal, dorsal tongue, and gingival swabs were subjected to mycobacterial polymerase chain reaction (PCR) analysis. RESULTS: A total of 67 subjects were enrolled in this study, 31 of whom underwent surgery due to strong clinical suspicion of NTM cervicofacial lymphadenitis. Positive genus and Mycobacterium avium specific PCR results were found in one patient from the control group, and in none of the patients with (suspected) NTM cervicofacial lymphadenitis. CONCLUSION: Our in-house routine diagnostic PCR assay on oral and oropharyngeal swabs was not suitable as a minimally invasive alternative to diagnostic sampling of excised lymph node material or punctate. It remains unclear whether the oral cavity and oropharynx might be a portal of entry for mycobacteria in NTM cervicofacial lymphadenitis.