Mycobacteria are classified into two groups, fast- and slow-growing. Often, fast-growing mycobacteria are assumed to have a higher metabolic activity than their slower counterparts, but in mature biofilms this assumption might not be correct. Indeed, when measuring the metabolic activity of mycobacterial biofilms with two independent non-invasive techniques (isothermal microcalorimetry and tunable diode laser absorption spectrometry), mature biofilms of slow- and fast-growing species appeared more alike than expected. Metabolic heat production rate was 2298 ± 181 µW for M. smegmatis and 792 ± 81 µW for M. phlei, while M. tuberculosis and M. bovis metabolic heat production rates were between these values. These small differences were further confirmed by similar oxygen consumption rates (3.3 ± 0.2 nMole/s and 1.7 ± 0.3 nMole/s for M. smegmatis and M. tuberculosis, respectively). These data suggest that the metabolic potential of slow-growing mycobacterial biofilms has been underestimated, particularly for pathogenic species.