ABSTRACT An epidemic of infections after video-assisted surgery (1,051 possible cases) caused by rapidly growing mycobacteria (RGM) and involving 63 hospitals in the state of Rio de Janeiro, Brazil, occurred between August 2006 and July 2007. One hundred ninety-seven cases were confirmed by positive acid-fast staining and/or culture techniques. Thirty-eight hospitals had cases confirmed by mycobacterial culture, with a total of 148 available isolates recovered from 146 patients. Most ( n = 144; 97.2%) isolates presented a PRA- hsp65 restriction pattern suggestive of Mycobacterium bolletii or Mycobacterium massiliense . Seventy-four of these isolates were further identified by hsp65 or rpoB partial sequencing, confirming the species identification as M. massiliense . Epidemic isolates showed susceptibility to amikacin (MIC at which 90% of the tested isolates are inhibited [MIC ₉₀ ], 8 μg/ml) and clarithromycin (MIC ₉₀ , 0.25 μg/ml) but resistance to ciprofloxacin (MIC ₉₀ , ≥32 μg/ml), cefoxitin (MIC ₉₀ , 128 μg/ml), and doxycycline (MIC ₉₀ , ≥64 μg/ml). Representative epidemic M. massiliense isolates that were randomly selected, including at least one isolate from each hospital where confirmed cases were detected, belonged to a single clone, as indicated by the analysis of pulsed-field gel electrophoresis (PFGE) patterns. They also had the same PFGE pattern as that previously observed in two outbreaks that occurred in other Brazilian cities; we designated this clone BRA100. All five BRA100 M. massiliense isolates tested presented consistent tolerance to 2% glutaraldehyde. This is the largest epidemic of postsurgical infections caused by RGM reported in the literature to date in Brazil.