In order to improve the recovery of mycobacteria from patients with cystic fibrosis, the present study evaluated a two-step decontamination procedure for clinical specimens. A total of 920 specimens obtained from 239 patients with cystic fibrosis were treated initially with N-acetyl-L-cysteine/sodium hydroxide. Of these specimens, 31 (3.3%) showed mycobacterial growth and 415 (45.1%) remained contaminated. Contaminated specimens were then subjected to a second round of decontamination, using a combination of N-acetyl-L-cysteine/sodium hydroxide and oxalic acid. Following this second decontamination, the number of specimens overgrown by microorganisms other than mycobacteria was reduced to 7.3%, and an additional 10 specimens positive for mycobacteria were found. The results suggest this two-step protocol could improve the recovery of mycobacteria from heavily contaminated specimens.