The diagnosis of extrapulmonary tuberculosis (EPTB) is challenging due to non-specific symptoms, invasive approach for specimen collection and most importantly, the paucibacillary status. The objective of this assay was to evaluate the efficacy of Myco/F lytic system, BACTEC Mycobacteria Growth Indicator Tube (MGIT) 960 system and Lowenstein-Jensen (L-J) medium for recovery of bacilli from sterile body fluids. 214 specimens (114 pleural fluid and 100 pus) from clinically diagnosed EPTB patients were collected and subjected to Ziehl-Neelsen (ZN) smear microscopy, L-J culture, MGIT 960 culture and Myco/F lytic culture.103 out of the 214 sterile body fluid samples yielded positive culture outcomes by any of the three methods. Among all the culture positive specimens, the recovery rate was 86.41% for Myco/F lytic, 75.73% for MGIT 960, and 42.72% for L-J medium. The mean time to positivity (TTP) was 27.06 ± 8.03 days for Myco/F lytic, 22.20 ± 7.84 days for MGIT960 and 42 ± 8.84 days for L-J medium. The rates of contamination were 6.54%, 3.74% and 2.80% for Myco/F lytic, MGIT960 and L-J medium respectively. Both Myco/F lytic and MGIT960 system were superior to L-J medium for recovery of bacilli from sterile body fluids. Myco/F lytic system was more favorable than MGIT960 regarding recovery rate and cost-effectiveness, thus can be considered as a promising alternative to MGIT960 system for diagnosing EPTB.