The pleuromutilin antibiotic lefamulin demonstrated activity against the most relevant bacterial pathogens causing sexually transmitted infections (STI), including (MIC, 0.02/0.04 mg/liter; = 15), susceptible and multidrug-resistant (MIC range, 0.002 to 0.063 mg/liter; = 6), and susceptible and resistant (MIC, 0.12/0.5 mg/liter; = 25). The results suggest that lefamulin could be a promising first-line antibiotic for the treatment of STI, particularly in populations with high rates of resistance to standard-of-care antibiotics.