We report the efficiency and cost-effectiveness of p-nitrobenzoic acid (PNB) testing in Middlebrook 7H10 agar medium for the identification of Mycobacterium tuberculosis complex (MTC). PNB-7H10 was compared with PNB-MGIT and BACTEC-NAP using 200 clinical mycobacterial isolates. PNB-7H10 showed 100% agreement with PNB-MGIT and BACTEC-NAP tests, and reduced the cost of PNB-MGIT test by 80%. PNB-7H10 agar is therefore an effective alternative to the costly PNB-MGIT and BACTEC-NAP tests, especially in resource-poor settings.