This study aims to evaluate the performance of a new vial (Myco/F Lytic) for the detection of mycobacteria from blood specimens. This vial is monitored in the BACTEC 9000 blood culture system. We compared it with the traditional method routinely used in our laboratory, which is a lysis-centrifugation based procedure. Of 275 samples tested in parallel by both methods, 23 from 20 patients grew mycobacteria (18 Mycobacterium avium complex, 4 M. tuberculosis and 1 M. simiae); 11 isolates were recovered using both systems, 12 were isolated with the Myco/F Lytic medium only, and none were isolated using the traditional method only (p < 0.05). Blood was the diagnostic sample for 12 patients with the Myco/F Lytic system and only 7 with the traditional system. The mean time to detection of mycobacteria with Myco/F Lytic medium was 17 days, whereas it was 44 days with the traditional method (p < 0.001). Identification by DNA probes was performed directly from the Myco/F Lytic bottle. Myco/F Lytic is a rapid, simple, safe and highly reliable diagnostic method for the detection of mycobacteria in blood.