Background: Topical dapsone gel is a sulfone antibiotic approved for acne treatment. No microbiology studies were conducted during dapsone gel clinical trials and it is unclear whether 1) dapsone has antimicrobial activity that may be of clinical relevance in dermatology and 2) dapsone could affect the normal microbiome of facial skin where it is most commonly applied. This study assessed the in vitro activity of dapsone versus Gram-positive and Gram-negative bacterial pathogens obtained from patients with infections. Methods: CANWARD is a national, annual, and ongoing surveillance system to assess the patterns of antibiotic-resistant pathogens in Canada. In 2014, 15 tertiary care medical centers collected 3,511 isolates from blood, respiratory tract, urine, and wounds. Antimicrobial susceptibility was assessed using CLSI broth microdilution method. Results: Dapsone demonstrated relatively poor activity versus Gram-negative bacilli with most MIC50, MIC90 in the range of 512μg/mL and >512μg/mL, respectively. In contrast, dapsone demonstrated activity versus Gram-positive cocci, such as Staphylococcus (including methicillin-resistant S. aureus [MRSA], methicillin-sensitive S. aureus [MSSA]), Streptococcus, and Enterococcus—several strains of S. epidermidis had MICs of 32 and 64μg/mL; there were strains of E. faecalis with MICs of 8, 16, 32, and 64μg/mL; and several strains of S. agalactiae and S. pyogenes demonstrated dapsone MICs of 4, 8, 16, 32, and 64μg/mL. Conclusion: Dapsone has demonstrated antimicrobial activity in vitro. Whether this activity is part of the mechanism of action of topical dapsone in acne remains unknown. There are limited cutaneous pharmacokinetic data with topical dapsone including skin concentrations achieved with topical dapsone therapy; however, topical dapsone as a 2% nanoemulsion has shown very high (1196-3837.34μg/cm2) local skin concentrations. At these high concentrations, topical dapsone would be expected to affect the skin flora of patients with acne (especially Gram-positive cocci, such as Staphylococcus and Streptococcus). These concentrations are multiple times higher (20x-1000x) than the dapsone MICs found for many MSSA, MRSA, S. epidermidis, S. agalactiae, and S. pyogenes, any of which may be present on the skin of acne patients. Whether this results in resistance to dapsone or more importantly results in resistance to chemically unrelated antimicrobials is currently unknown.