Introduction: Aseptic procedures are undertaken by nurses in the general ward. The lack of nurse's aseptic techniques in clinical setting result to patients or healthcare expositions to hospital-acquired infections which are most of the time caused by lack of knowledge or ignorance of implementing the principles of aseptic technique. This study aims to assess nurses' knowledge and the possible barriers in the implementation of the principles of asepsis in healthcare setting. Methods: A cross-sectional study design was used to assess the nurses' knowledge and the possible barriers for implementing principles of asepsis in healthcare setting among. The study was carried out at Clinique Van Norm (CVN), a free Methodist Church related institution located in the north District of Bujumbura city in Burundi, from October to November, 2021 among nurses who work within three services of the clinic (Gynecology and Obstetrics, Pediatric and Operating room). A convenience sampling method was used to invite all nurses (44) working in the 3 services of CVN to participate in our study. A self-report method involving questionnaire completion with three components addressing demographic data, participants' knowledge and barriers to implement the principles of asepsis was used to collect data which were later on analyzed by SPSS version 21. Results: Research findings show a significant participant's poor knowledge on principles of asepsis in the hospital as for almost variables used to assess their knowledge, they scored less than 50% except for the time of using sterile gloves in which most of the participants (71.4%) said that they do use sterile gloves when indwelling urinary catheters, labor and delivery, newborn care, wound dressing or suturing, or any time handling aseptic equipment, inserting nasogastric feeding tube...