The rate of recovery and time to the detection of mycobacteria in clinical specimens were measured in traditional egg-based media cultures and on Middlebrook 7H11 agar plate cultures using microcolony detection. In the 5438 specimens processed, a total of 293 (5.4%) clinically relevant mycobacterial isolates were detected (Mycobacterium tuberculosis, n = 231; Mycobacterium avium complex, n = 60; Mycobacterium kansasii, n = 2). Of these, 227 (77%) and 237 (81%) isolates were detected on Lowenstein-Jensen medium and Coletsos medium, respectively, and 265 (90%) isolates were detected on Middlebrook 7H11 plates examined microscopically. The detection time was shorter with the microcolony detection method. While the Lowenstein-Jensen and Coletsos media required an average of 23 and 25 days, respectively, for first detection of mycobacteria, microcolony detection on Middlebrook 7H11 required an average of only 12 days. For acid-fast, stain-positive specimens that were culture positive for Mycobacterium tuberculosis, the average interval to positivity was nine days for the microcolony method compared with 20 and 21 days for the Lowenstein-Jensen and Coletsos media, respectively. Microscopic detection on Middlebrook 7H11 agar plates is a rapid, accurate and inexpensive method of detecting Mycobacterium tuberculosis and other clinically important mycobacteria.