Mycobacterium neoaurum is a saprophytic, soil-dwelling bacterium. The strain NRRL B-3805 converts phytosterols to androst-4-ene-3,17-dione (androstenedione; AD), a precursor of multiple C19 steroids of importance to industry. NRRL B-3805 itself is able to convert AD to other steroid products, including testosterone (Ts) and androst-1,4-diene-3,17-dione (androstadienedione; ADD). However to improve this strain for industrial use, genetic modification is a priority. In this chapter, we describe a range of genetic techniques that can be used for M. neoaurum NRRL B-3805. Methods for transformation, expression, and gene knockouts are presented as well as plasmid maintenance and stability.