Background Decentralizing childhood tuberculosis services, including diagnosis, is now recommended by the WHO and could contribute to increasing tuberculosis detection in high burden countries. However, implementing microbiological tests and clinical evaluation could be challenging for health care workers (HCWs) in Primary Health Centers (PHCs) and even District Hospitals (DHs). We sought to assess the acceptability of decentralizing a comprehensive childhood tuberculosis diagnosis package from HCWs’ perspective. Design/Methods We conducted implementation research nested within the TB-Speed Decentralization study. HCWs from two health districts of Cambodia, Cameroon, Côte d’Ivoire, Mozambique, Sierra Leone, and Uganda implemented systematic screening, nasopharyngeal aspirates (NPA) and stool sample collection with molecular testing, clinical evaluation and chest X-ray (CXR) interpretation. We investigated their experiences and perceptions in delivering the diagnostic package components in 2020-21 using individual semi-structured interviews. We conducted thematic analysis, supported by the Theoretical Framework of Acceptability. Results HCWs (n=130, 55% female, median age 36 years, 53% nurses, 72% PHC-based) perceived that systematic screening, although increasing workload, was beneficial as it improved childhood tuberculosis awareness. Most HCWs shared satisfaction and confidence in performing NPA, despite procedure duration, need to involve parents/colleagues and discomfort for children. HCWs shared positive attitudes towards stool sample-collection but were frustrated by delayed collection associated with cultural practices, transport and distance challenges. Molecular testing, conducted by nurses or laboratory technicians, was perceived as providing quality results, contributing to diagnosis. Clinical evaluation and diagnosis raised self-efficacy issues and need for continuous training and clinical mentoring. HCWs valued CXR, however complained that technical and logistical problems limited access to digital reports. Referral from PHC to DH was experienced as burdensome. Conclusion HCWs at DH and PHC-levels perceived and experienced decentralized childhood tuberculosis diagnosis as acceptable. Implementation however could be hampered by feasibility issues, and calls for innovative referral mechanisms for patients, samples and CXR.