Background and rationale: Infections caused by bacteria are significant public health problems and the roots of much illness and death in Laos. Hemoculture is the gold standard for the diagnosis of septicemia and the aetiological agents. However, the result of blood culture depend on many factors, from the collection of blood by nurses until completing and reporting the laboratory analysis. The process of blood collection by nurses plays an important role for optimizing culture yields in the laboratory and the result will be more accurate with a lower contamination rate, if performed using strict sterilized techniques. Correct blood collection and culture practices will also reduce the risks of needle-stick injuries and harm. Objective: To study behavior on blood collection from patients by nurses for culture at Microbiology Laboratory of Mahosot Hospital, Vientiane Capital. Methodology: The study was descriptive, cross-sectional quantitative study to the nurses who were performing blood collection for culture and who answered questionnaires. The reported behavior of the nurses was also compared to that observed by the researcher. Data entered into the Excel and analyzed with STATA. Results: The majority of the study nurses were female (92%) with a mean age of 33 years, a mid-level of nursing study (82.4%), and with an average duration of working of 7 years. The awareness of nurses about the importance of blood collection technique for culture was in the range from 43.3% and 30.0% rating as high and intermediate respectively. Right attitude towards accurate practices was 27.0%. The reported correct practice of blood collection by the nurses was 38% however the correspondent figure by observation was only 12%. Older age, longer work experience, better work knowledge, attitude were significantly associated with correct blood collection procedures (P-value <0.05). Conclusion The techniques and knowledge of the nurses for collecting blood cultures remains low due to lack of knowledge, techniques, procedures and methods to collect and deliver samples. Therefore, adequate and regular trainings are urgently needed.