Extra-pulmonary mycobacterial infections are characterized by a paucibacillary nature and extra-pulmonary samples consist of different matrices; the processing of these samples requires a high level of manual skills and non-standardized procedures. The aim of this study was to compare the performance of MYCO-TB with MycoPrep on extra-pulmonary samples in terms of Mycobacteria detection, culture contamination and suitability for molecular assay. This prospective study was conducted on 201 extra-pulmonary samples from suspected cases of mycobacterial infection. Specimens were divided into two equal aliquots; one was decontaminated with MYCO-TB the other with MycoPrep. The contamination rate of liquid cultures was significantly different: 2.5% (5/201) for MYCO-TB and 7.5% (15/201) for MycoPrep (p = 0.036). At least 1 Mycobacterium tuberculosis complex (MTBc) positive culture was detected in 6 specimens treated with MYCO-TB and 8with MycoPrep, without significant differences in times to positivity (TTP) in liquid culture. No Xpert MTB/RIF Ultra invalid results were obtained with samples decontaminated with MYCO-TB. The MYCO-TB kit had greater activity than MycoPrep in the digestion and decontamination of extra-pulmonary specimens for the detection of Mycobacteria, supporting the use of MYCO-TB in this type of sample. Ready-to use reagents, rapid protocol and single-sample formulation of MYCO-TB reduced the level of manual skills required as well as the risk of sample contamination.