Avian mycobacteriosis (AM) is a significant disease of birds in the Taronga Zoo (TZ) collection. This study was designed to determine which species of mycobacteria were responsible for AM; and to describe the epidemiological and pathological features of AM at TZ. The study applied multiple molecular techniques (PCR, qPCR) on tissue samples (2007-2013) from 102 birds, and revealed that Mycobacterium genavense (MG) and M. avium (MA) are the two most significant organisms associated with AM at TZ. Molecular techniques significantly improved the detection and speciation of Mycobacterium spp. in tissues, particularly of MG which had a 30-fold increased detection and speciation rate. Eighty-one birds were confirmed to have AM at TZ between 2007-2013 and the prevalence was 10.01% - the highest documented in a zoo in the English literature. In total, 47 birds were infected with MG, 35 with MA, and two with M. terrae. Nine birds were infected with more than one mycobacterial species. Thirty-eight species and nine orders were affected. Columbiformes were overrepresented and psittaciformes underrepresented. Birds with AM came from 30 different enclosures, and all but one enclosure had both MA and MG infections. Enclosure disinfection reduced the prevalence of AM in four of the five treated enclosures. Epidemiologically, MA and MG behaved remarkably similarly. Pathological features of disease in these 81 birds were analysed. The most commonly affected organs were the liver (45.6% of birds), lungs (37%), spleen (19.8%), and small intestine (19.8%). MG was significantly more likely to occur in the heart and the kidney than MA. 26% of birds had detectable pathology in the alimentary tract, 37% in the respiratory system, and 47.0% in the liver. MA infected birds were more likely to form morphologically tuberculous lesions, whereas MG infected birds were more likely to form nontuberculous (lepromatous) lesions. 81.5% of birds had comorbid pathological conditions.