Infections associated with Mycobacterium haemophilum are underdiagnosed because specific culture methods required for its recovery are not applied routinely. Using polymerase chain reaction (PCR) technology on fine needle aspirates and biopsied specimens from 89 children with cervicofacial lymphadenitis, we assessed the importance of M. haemophilum. Application of a Mycobacterium genus-specific real-time PCR in combination with amplicon sequencing and a M. haemophilum-specific PCR resulted in the recognition of M. haemophilum as the causative agent in 16 (18%) children with cervicofacial lymphadenitis. M. avium was the most frequently found species (56%), and M. haemophilum was the second most commonly recognized pathogen. Real-time PCR results were superior to culture because only 9 (56%) of the 16 diagnosed M. haemophilum infections were positive by culture.