Methods are disclosed herein for treating an infection with a Mycobacterium infection. Methods are also disclosed for treating a Mycobacterium biofilm. The method include the use of trehalose dimycolate hydrolase, a variant thereof, or a nucleic acid encoding trehalose dimycolate hydrolase or a variant thereof Methods are also disclosed for detecting a Mycobacterium in a sample from a subject. The methods include treating the sample with trehalose dimycolate hydrolase or a variant thereof, and detecting a Mycobacterium nucleic acid in the sample.