Antimicrobial resistance and the mechanisms implicated were studied in 148 clinical Enterococcus faecium isolates with a quinupristin-dalfopristin (Q/D) MIC > or =1 microg/mL. As expected, higher levels of resistance were detected for macrolide antibiotics (94% erythromycin, 100% azithromycin, 100% josamycin). High-level resistance to gentamicin and streptomycin was detected in 18.9% and 66.2% of isolates, respectively, in our series of enterococci. Resistance against tetracycline was found in only 12.8% of the isolates, and 13 isolates were resistant to vancomycin. The dalvabancin MIC(90) for these isolates was >16 microg/mL. Polymerase chain reaction screening for the previously described streptogramin resistance determinants erm(A), erm(B), mefA/E, vat(D), and vat(E) was performed to determine the prevalence of streptogramin resistance mechanisms in these clinical enterococcal isolates. The combination of erm(B) and vat(D) genes encoding resistance to streptogramins was detected in 1 Q/D-resistant E. faecium isolate. A total of 131 Q/D-nonsusceptible enterococci only contained the erm(B) gene. The erm(B) promoter sequence reveals no differences between the strains analyzed, regardless of the Q/D MIC value.