The versatility of denaturing gradient gel electrophoresis (DGGE) protocol provides enough grounds for its wide application over an array of microorganisms. This work was designed to evaluate DGGE for the detection and confirmation of mycobacteria and their association, if any, with waterborne pathogens. A total of 76 samples comprising raw untreated water, schmutzdecke, floccules and final treated water obtained from a common water source, and two water treatment works (WTW1 and WTW2), were analysed. Thirty-five species were identified from the overall samples, with 7% (5/76), 13% (10/76) and 26% (20/76) from the common raw water source, WTW1 and WTW2 respectively. The majority of the species were Cyanobacteria, with high dominance in the raw water entering WTW2. In the final treated water of WTW1 Eutreptiella braarudii was found, and that of WTW2 contained Anabaena nereformis, Anabaena torulosa and Podocarpus nerrifolius. Furthermore, one Mycobacterium species was found in the raw water of WTW1 aside from the detection of Mycobacterium avium ssp. paratuberculosis by the technique. No association between mycobacteria and the other species was observed. This implies DGGE may be employed to study the diversity of other akin mycobacterial species from various sources, and not as a direct means of elucidating microbial associations.