Mycobacterium brisbanense is a member of Mycobacterium fortuitum third biovariant complex, which includes rapidly growing Mycobacterium spp. that normally inhabit soil, dust and water, and can sometimes cause respiratory tract infections in humans. We present the first whole-genome analysis of M. brisbanense UM-WWY which was isolated from a 70-year-old Malaysian patient. Molecular phylogenetic analyses confirmed the identification of this strain as M. brisbanense and showed that it has an unusually large genome compared with related mycobacteria. The large genome size of M. brisbanense UM-WWY (∼7.7Mbp) is consistent with further findings that this strain has a highly variable genome structure that contains many putative horizontally transferred genomic islands and prophage. Comparative analysis showed that M. brisbanense UM-WWY is the only Mycobacterium species that possesses a complete set of genes encoding enzymes involved in the urea cycle, suggesting that this soil bacterium is able to synthesize urea for use as plant fertilizers. It is likely that M. brisbanense UM-WWY is adapted to live in soil as its primary habitat since the genome contains many genes associated with nitrogen metabolism. Nevertheless, a large number of predicted virulence genes were identified in M. brisbanense UM-WWY that are mostly shared with well-studied mycobacterial pathogens such as Mycobacterium tuberculosis and Mycobacterium abscessus. These findings are consistent with the role of M. brisbanense as an opportunistic pathogen of humans. The whole-genome study of UM-WWY has provided the basis for future work of M. brisbanense.