Introduction In 2007, a new federal legislation in Belgium prohibited non-biosafety level 3 laboratories to process culture tubes suspected of containing mycobacteria. Aim To present mycobacterial surveillance/diagnosis data from the Belgian National Reference Centre for mycobacteria (NRC) from 2007 to 2016. Methods This retrospective observational study investigated the numbers of analyses at the NRC and false positive cultures (interpreted as containing mycobacteria at referring clinical laboratories, but with no mycobacterial DNA detected by PCR in the NRC). We reviewed mycobacterial species identified and assessed trends over time of proportions of nontuberculous mycobacteria (NTM) vs Mycobacterium tuberculosis complex (MTBc), and false positive cultures vs NTM. Results From 2007 to 2016, analyses requests to the NRC doubled from 12.6 to 25.3 per 100,000 inhabitants. A small but significant increase occurred in NTM vs MTBc proportions, from 57.9% (587/1,014) to 60.3% (867/1,437) (p < 0.001). Although NTM infection notification is not mandatory in Belgium, we annually received up to 8.6 NTM per 100,000 inhabitants. M. avium predominated (ca 20% of NTM cultures), but M. intracellulare culture numbers rose significantly, from 13.0% (74/587) of NTM cultures in 2007 to 21.0% (178/867) in 2016 (RR: 1.05; 95% CI: 1.03–1.07). The number of false positive cultures also increased, reaching 43.3% (1,097/2,534) of all samples in 2016. Conclusion We recommend inclusion of NTM in sentinel programmes. The large increase of false positive cultures is hypothesised to result from processing issues prior to arrival at the NRC, highlighting the importance of sample decontamination/transport and equipment calibration in peripheral laboratories.