Linezolid is an antimicrobial agent for the treatment of multiresistant Gram-positive infections. We assessed the impact of linezolid on the microbiota and the emergence of resistance and investigated its relationship with plasma pharmacokinetics of the antibiotic. Twenty-eight patients were treated for the first time with linezolid administered orally (n = 17) or parenterally (n = 11) at 600 mg twice a day. Linezolid plasma pharmacokinetic analysis was performed on day 7. Colonization by fecal enterococci, pharyngeal streptococci, and nasal staphylococci were assessed using selective media with or without supplemental linezolid. The resistance to linezolid was characterized. The treatment led to a decrease of enterococci, staphylococci, and streptococci in the fecal (P = 0.03), nasal, and pharyngeal (P < 0.01) microbiotas. The appearance of resistant strains was observed only in enterococci from the fecal microbiota between the 7th and 21st days of treatment in four patients (14.3%). The resistance was mainly due for the first time to the mutation G2447T in the 23S rRNA gene. No pharmacokinetic parameters were significantly different between the patients, regardless of the appearance of resistance. The emergence of linezolid resistance during treatment was observed only in the intestinal microbiota and unrelated to pharmacokinetic parameters. However, colonization by Gram-positive bacteria was reduced as a result of treatment in all microbiotas.