Mycobacteria naturally aggregate in water, a characteristic that may serve to protect them against disinfection in wastewater. Secondary effluent was spiked with Mycobacterium terrae (M. terrae), sequentially filtered through 100-, 41-, and 20-microm nylon filters to partition aggregate sizes, confirmed using particle-size analysis and microscopy. Each sample was exposed to doses of UV light (10 to 60 mJ/cm2 at 254 nm) and free chlorine (27 to 150 mg-min/L at 4 degrees C). Inactivation of M. terrae in wastewater was initially rapid, with 2.5 log reduction at 14 mJ/cm2 and 56 mg-min/L for UV and free chlorine, respectively. However, in effluent and 100-microm filtered wastewater, spiked M. terrae was present to the highest doses evaluated. Interestingly, M. terrae passed through 41- and 20-microm filters were inactivated rapidly, with no survivors after moderate disinfection doses. Inactivation of Mycobacteria in wastewater may be compromised by aggregates larger than 41 microns.