Mycobacterium smegmatis is a rapidly growing environmental species not considered a human pathogen. We identified 22 human isolates of M. smegmatis from Australia and the southern United States: 19 were from skin or soft-tissue infections, and none were from urine or the male genital tract. These isolates closely resembled Mycobacterium fortuitum, except for a negative three-day arylsulfatase test; growth at 43-45 C; a low semiquantitative catalase test; and, in 50% of isolates, a late-developing, yellow-to-orange pigment. The isolates were biochemically identical to four reference strains and the type strain of M. smegmatis. Isolates were resistant to isoniazid and rifampin but susceptible to ethambutol, doxycycline, sulfamethoxazole, ciprofloxacin, imipenem, and amikacin. Eleven patients treated on the basis of in vitro susceptibility tests responded well to therapy. The similarity of M. smegmatis to M. fortuitum and the failure to recognize that the former is an environmental species may have contributed to previous failures to recognize it as a human pathogen.