With the widespread use of antiseptics in healthcare facilities for the prevention of methicillin-resistant Staphylococcus aureus (MRSA) transmission, there are concerns for antiseptic tolerance and resistance. We sought to understand the use of chlorhexidine and octenidine, carriage of qac genes, and reduced antiseptic susceptibilities. A serial cross-sectional study was conducted in an acute care hospital and three extended-care facilities of a healthcare network in June–July, 2014–2016. Two of the extended-care facilities were exposed to intranasal octenidine and universal daily chlorhexidine/octenidine bathing. The minimum inhibitory concentration (MIC) levels and qac genes were determined by broth microdilution tests and whole genome sequencing respectively. Multivariable logistic regression was used to assess for the independent associations between antiseptic exposures, qac genes, and reduced antiseptic susceptibilities. A total of 878 MRSA isolates were obtained. There were associations between qacA/B carriage and chlorhexidine (adjusted odds ratio (aOR) 7.80; 95% confidence interval (CI) 3.25–18.71) and octenidine (aOR 11.79; 95% CI 5.14–27.04) exposures. Chlorhexidine exposure was associated with reduced chlorhexidine susceptibility (MIC ≥4 mg/L) (aOR 3.15; 95% CI 1.14–8.74). Carriage of qacA/B (aOR 10.65; 95% CI 4.14–27.40) or qacC (aOR 2.55; 95% CI 1.22–5.32) had an association with reduced chlorhexidine susceptibility; while MRSA sequence type modified the association. However, we found no direct association between (i) antiseptics use and qacC carriage, (ii) octenidine exposure and reduced susceptibility, and (iii) reduced octenidine susceptibility and qacA/B or qacC carriage. Antiseptic exposures were associated with carriage of qac genes. Chlorhexidine exposure was associated with reduced chlorhexidine susceptibility, requiring continued surveillance for the emergence of resistance.