Background: Prevention of central-line–associated bloodstream infections (CLABSIs) and methicillin-resistant Staphylococcus aureus (MRSA) infections requires a multifaceted approach including strategies to decrease cutaneous bacterial colonization. Prior studies have shown benefit from chlorhexidine-gluconate (CHG) skin application on CLABSI and MRSA infection rates in intensive care units (ICUs); however, the use of CHG in the non-ICU population has not been well studied. Methods: We performed a quasi-experimental before-and-after study to evaluate the use of daily 2% CHG wipes in non-ICU patients at a 1,000 bed acute-care teaching hospital beginning in November 2017. The study population included adult and pediatric patients with central venous catheters on non-ICU units, excluding patients on the following units: stem cell transplant and hematologic malignancy (these units had already established use of CHG skin application as a standard prior to the intervention), labor and delivery, and psychiatry. CHG was applied according to the manufacturer’s instruction by nurses or nurse aides and random monthly auditing of compliance was performed. NHSN CLABSI, hospital-onset MRSA bacteremia, and hospital-onset MRSA LabID rates were compared for the period 24 months before the intervention (November 1, 2015, through October 31, 2017) to the 24-month period after the intervention (November 1, 2017, through October 31, 2019) using a paired t test. Notably, the health system also discontinued the use of contact precautions for patients with MRSA (excluding MRSA from open, draining wounds) 11 months prior to onset of this intervention. Results: The CLABSI rate decreased by 26% from 0.594 events per 1,000 central-line days (n = 50) before the intervention to 0.438 events per 1,000 central-line days (n = 38) after the intervention (P = 0.19). The number of CLABSIs with gram-positive organisms also decreased by 29%. MRSA LabID rates decreased by 37% from 0.301 events per 1,000 patient days (n = 119) to 0.189 events per 1,000 patient days (n = 75) (P = 0.01). MRSA bacteremia rates decreased by 79% from 0.058 events per 1,000 patient days (n = 23) to 0.012 events per 1,000 patient days (n = 5) (P < 0.01). Compliance with the intervention was 83% (n = 225). Conclusions: Daily CHG skin application in non-ICU patients with central venous catheters is an effective strategy to prevent CLABSIs and MRSA infections. We observed a decrease in MRSA LabID and bacteremia rates despite discontinuation of contact precautions. These findings suggest that a horizontal prevention approach of daily CHG skin application may be an effective alternative to contact isolation to interrupt transmission of MRSA in hospitalized patients outside the ICU setting.