BACKGROUND: Environmental cleaning is a key infection prevention and control (IPC) intervention in healthcare settings. The U.S. Centers for Disease Control and Prevention (CDC), with Infection Control Africa Network (ICAN), developed best practices for global healthcare environmental cleaning in resource-limited settings to help fill gaps in guidance in low- and middle-income countries (LMICs). We aimed to evaluate the feasibility, appropriateness, and acceptability of a quality improvement toolkit developed to assist with implementing the CDC/ICAN best practices at Lagos University Teaching Hospital in Nigeria. METHODS: A mixed-methods approach was used to evaluate the implementation of the toolkit from March through September of 2021. A monitoring checklist assessed feasibility after three defined steps within the toolkit. Key informant interviews and electronic surveys were conducted with toolkit team members at three time points during implementation to assess appropriateness and acceptability. A deductive analytic process was used to code and analyze interview data based on constructs of appropriateness and acceptability. Additional codes and sub-themes that emerged during analysis followed an inductive process. RESULTS: Within the interviews and surveys, themes identified for the appropriateness included concern related to (1) time commitment for the toolkit activities and (2) resources required to sustain improvements. Themes identified for acceptability included (1) perceived challenges with time commitment and resource requirements, (2) perceived effectiveness of toolkit structure and usability, (3) perceived benefits and success associated with knowledge gained about environmental cleaning and environmental cleaning staff, (4) perceived benefits and success associated with the training for cleaning staff undertaken during toolkit implementation, and (5) perceived benefits and success associated with the multidisciplinary team approach with the inclusion of facility leadership and a project coordinator. CONCLUSIONS: The results showed that the toolkit materials were feasible within the local context and highlighted perceived effectiveness, benefits, and success of the toolkit process and experience contributing to a high level of acceptability. Challenges relating to time commitment and concern for sustainability have implications for the appropriateness of this toolkit, similar approaches to quality improvement, and the need for strengthening support for IPC improvements at the facility and national levels in resource-limited healthcare settings in LMICs.