Background. Nasal decolonization procedures against the opportunistic pathogen Staphylococcus aureus rely on topical antimicrobial drug usage, whose impact on the nasal microbiota is poorly understood. We examined this impact in healthy S. aureus carriers and noncarriers. Methods. This is a prospective interventional cohort study of 8 S. aureus carriers and 8 noncarriers treated with nasal mupirocin and chlorhexidine bath. Sequential nasal swabs were taken over 6 months. S. aureus was detected by quantitative culture and genotyped using spa typing. RNA-based 16S species-level metabarcoding was used to assess the living microbial diversity. Results. The species Dolosigranulum pigrum, Moraxella nonliquefaciens and Corynebacterium propinquum correlated negatively with S. aureus carriage. Mupirocin treatment effectively eliminated S. aureus, D. pigrum and M. nonliquefaciens, but not corynebacteria. S. aureus recolonization in carriers occurred more rapidly than recolonization by the dominant species in noncarriers (median 3 vs. 6 months, respectively). Recolonizing S. aureus isolates had the same spa type as the initial isolate. Conclusions. Mupirocin-chlorhexidine treatment had a long-lasting impact on the nasal microbiota. S. aureus recolonization predated microbiota recovery, emphasizing the strong adaptation of this pathogen to the nasal niche and the transient efficacy of the decolonization procedure.