BackgroundCatheter-associated urinary tract infection is a global problem but it can be prevented with the appropriate implementation of evidence-based guidelines. This study was conducted to assess the level of compliance of healthcare workers with the catheter-associated urinary tract infection prevention guidelines during the insertion of a urinary catheter.MethodsAn observational study using a descriptive cross-sectional design was conducted at Sana’a City hospitals, Yemen. All the nurses and physicians from the governmental, teaching, and private hospitals were eligible to participate in the study. The data collection was performed through convenience sampling from March 2020 to December 2020, using a structured observational checklist prepared specifically for this study.ResultsThe majority of the urinary catheter insertions were performed by nurses. There were no written policy or procedures for an urinary catheter insertion and no in-service education or training departments in the majority of the hospitals. The overall mean score of compliance was 7.31 of 10. About 71% of the healthcare workers had a high or acceptable level of compliance and 29% had an unsafe level of compliance. Compliance was low for maintaining aseptic technique throughout the insertion procedure, using a single use packet of lubricant jelly, performing hand hygiene immediately before insertion, and securing the urinary catheter once inserted. Factors affecting the healthcare workers compliance were gender, the working ward/unit of the healthcare workers, the availability of a written policy/procedure and a department or unit for in-service education.ConclusionYemeni healthcare workers’ overall compliance was acceptable but it was unsafe in several critical measures. There is an urgent need for developing, implementing, and monitoring national guidelines and institutional policy and procedures for catheter-associated urinary tract infection prevention. Periodical in-service education and training programs and adequate access to the necessary materials and supplies are paramount.