Mycobacterium simiae is an emerging and spreading pathogen in Iran and little data about its drug susceptibility test (DST) and no standard treatment regimen are available. We report a case of multidrug-resistant M. simiae respiratory infection in a 65-year-old woman with a history of previous Mycobacterium tuberculosis infection. The patient was treated with clarithromycin, levofloxacin, and cotrimoxazole for one year and eventually died while still suffering from respiratory problems. For DST, broth microdilution method was used according to the Clinical and Laboratory Standards Institute guidelines as well as molecular DST in clinical isolate. Mycobacterium simiae was resistant to streptomycin, moxifloxacin, clarithromycin, and cotrimoxazole antibiotics and was sensitive to clofazimine and amikacin antibiotics. Inappropriate use of antibiotics without determining the pattern of antibiotic resistance increases the likelihood of resistance and, for resistant specimens, the need to review the treatment protocol and replace antibiotics. Effectiveness based on antibiotic resistance pattern is essential.