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OBJECTIVES The objective of this study was to determine the in vitro activity of iclaprim and comparator agents against 7618 Gram-positive clinical isolates collected in the periods 2004-2006, 2012-2014 and 2015-2016. METHODS Antimicrobial susceptibility testing was performed by the broth microdilution method and the minimum inhibitory concentrations (MICs) were interpreted according to Clinical and Laboratory Standards Institute (CLSI) guidelines. RESULTS Iclaprim MIC50/MIC90 values were 0.06/0.12μg/mL for Staphylococcus aureus, including methicillin-susceptible and methicillin-resistant strains, and 0.015/0.03, 0.12/0.5 and 0.03/0.06μg/mL, respectively, for Streptococcus pyogenes, Streptococcus agalactiae and Streptococcus dysgalactiae over 8 years within the period from 2004 to 2016. Iclaprim was 8-32-fold more potent than trimethoprim. Against S. aureus, including methicillin-resistant strains, iclaprim was more active than standard-of-care intravenous antibiotics used to treat Gram-positive skin infections. Iclaprim was up to 16-fold more potent than vancomycin and linezolid and was 4-8-fold more potent than daptomycin. CONCLUSIONS Iclaprim demonstrated potent and consistent activity among Gram-positive clinical isolates collected globally between 2004 and 2016.