Background: The skin is the largest organ of the body, where many microorganisms reside. These microorganisms can produce detrimental effects if introduced into the body through surgical incisions. Therefore, the aseptic applications of the surgical site, with proper antiseptics, are necessary prior to an invasive medical procedure. Objectives: This study aims to identify the knowledge, attitudes, and practices on skin asepsis with the use of antiseptics and to identify the most preferred antiseptic by healthcare professionals in Sri Lanka, which allows understanding of whether the current procedure of skin antisepsis is up to the standard. Methods: A descriptive cross-sectional study was carried out among 210 healthcare professionals. Results: Most participants had poor knowledge (92.9%) and moderate attitudes with moderate practices (55.2%). Conclusion: This study highlights the need to improve further the existing skin antiseptic applications to address the gaps in knowledge, attitudes, and practices.