Background: The most common means of transmission of Healthcare-Associated Infections (HAIs) is the contaminated hand of Healthcare Workers (HCWs), proper handwashing by HCWs is the best method of halting this transmission, however, there is poor adherence to the practice of the simple handwashing procedure in many health facilities. Aims: To assess the knowledge and practice of proper handwashing, to identify the factors impeding adherence to its good practice among HCWs and to identify the handwashing and hand drying facilities available to these HCWs. Study Design: This is a descriptive cross-sectional study. Place and Duration of Study: The study was carried out at Federal Teaching Hospital, Ido-Ekiti (FETHI), Southwestern Nigeria between July 2020 and December 2020. Methods: The study involved 328 randomly selected HCWs. Data was collected using pretested self administered questionnaires, entered into Microsoft Excel 2016 and analyzed by SPSS version 23. Results: Only 31.10% of respondents had good knowledge of handwashing while 80.18% had good practice of handwashing. Longer stay in service and being a doctor or nurse were associated with good knowledge and good practice of handwashing. Only 16.46% of respondents had previous training on handwashing within 3 years prior to this study. Previous training on handwashing was associated with good knowledge of handwashing (P=.001). Up to 68.60% of respondents used stored water and soap for handwashing. The most common hand drying facility reported in the study was common towel, no respondent ever used paper towel. The most common reason given for poor adherence to handwashing practices was the busy schedule. Conclusion: Hospital managers must prioritize the regular provision of handwashing and hand drying facilities for their centre, in addition to organizing regular hand hygiene training for HCWs, to improve their knowledge and practice of handwashing and thus reduce the rate of HAIs