Background Reducing the transmission of pathogens from medical devices continues to be a focus of the healthcare industry. High-level disinfection (HLD) is one method frequently employed to eliminate disease-causing microorganisms. HLD is a complex process that requires meticulous cleaning and attention to numerous details such as temperature, concentration of disinfectant, and exposure time among others. Staff must be properly trained and competent to ensure the process is being performed correctly. During a review of processes, it was noted that practices were inconsistent between like departments. Therefore, an audit program was developed and implemented to improve consistency, ensure best practices, and provide support to department staff. Methods A detailed review of industry recommendations, regulatory standards, and manufacturer's instructions for use was used to facilitate and inform the development of auditing tools. Audits focused on key departments such as Endoscopy, Transesophageal Echocardiogram (TEE), Ultrasound (US), as well as areas using liquid high-level disinfectants (LHLD). A compliance-based audit schedule was designed to ensure routine monitoring of processes. Audit findings were labeled as general or critical, with each requiring action plan development and follow-up from department leadership. Results The audit program has proven beneficial in highlighting deviations in practice, enhancing communication, and allowing the team to address real or perceived barriers. Pre-audit compliance averaged 87.7% (US), 88.1% (TEE), 91.2% (Endoscopy), and 94.5% (LHLD). Six months post implementation, overall compliance had increased to an average of 95.2% (US), 94.4% (TEE), 96.8% (Endoscopy), and 96% (LHLD) respectively. Conclusions Implementation of a routine audit program with actionable follow-up increased overall compliance for departments utilizing high-level disinfection while providing a tool for sustainability. An audit program is beneficial to ensure safe patient care by staying current and compliant on HLD standards, recommendations, and best practices. Reducing the transmission of pathogens from medical devices continues to be a focus of the healthcare industry. High-level disinfection (HLD) is one method frequently employed to eliminate disease-causing microorganisms. HLD is a complex process that requires meticulous cleaning and attention to numerous details such as temperature, concentration of disinfectant, and exposure time among others. Staff must be properly trained and competent to ensure the process is being performed correctly. During a review of processes, it was noted that practices were inconsistent between like departments. Therefore, an audit program was developed and implemented to improve consistency, ensure best practices, and provide support to department staff. A detailed review of industry recommendations, regulatory standards, and manufacturer's instructions for use was used to facilitate and inform the development of auditing tools. Audits focused on key departments such as Endoscopy, Transesophageal Echocardiogram (TEE), Ultrasound (US), as well as areas using liquid high-level disinfectants (LHLD). A compliance-based audit schedule was designed to ensure routine monitoring of processes. Audit findings were labeled as general or critical, with each requiring action plan development and follow-up from department leadership. The audit program has proven beneficial in highlighting deviations in practice, enhancing communication, and allowing the team to address real or perceived barriers. Pre-audit compliance averaged 87.7% (US), 88.1% (TEE), 91.2% (Endoscopy), and 94.5% (LHLD). Six months post implementation, overall compliance had increased to an average of 95.2% (US), 94.4% (TEE), 96.8% (Endoscopy), and 96% (LHLD) respectively. Implementation of a routine audit program with actionable follow-up increased overall compliance for departments utilizing high-level disinfection while providing a tool for sustainability. An audit program is beneficial to ensure safe patient care by staying current and compliant on HLD standards, recommendations, and best practices.