Background: : Antimicrobial resistance (AMR) presents an escalating global health concern. Medical students, as future prescribers and stewards of antimicrobials, play a critical role in combating AMR. However, inappropriate antibiotic use, such as unnecessary continuation of prophylaxis for common colds, persists among practitioners. Resistant infections can prolong treatment duration and increase mortality risk, particularly in immunocompromised patients. Objective: : To evaluate the knowledge and attitudes of sixth year medical students regarding antibiotic use before and after exposure to the Lao adult and pediatric Antimicrobial Prescribing guidelines. Methodology: : A before and after study was conducted using a structured questionnaire administered via face-to-face interviews to test student knowledge. Students were exposed to prescribing guidelines by the percentage of students with high or low knowledge scores before and after the intervention were compared. Results: : A total of 134 students participated; 52.2% were female, with a mean age of 20.78 years. Most students (82.8%) had completed high school, and 61.2% resided in Vientiane capital. Pre-intervention assessment revealed that 72.4% of students demonstrated low knowledge of adult antimicrobial prescribing, which improved post intervention, with 65.7% achieving a high knowledge level. Similarly, for pediatrics antimicrobial prescribing, 73.1% exhibited low knowledge pre-intervention, improving to 56.7% with high knowledge post-intervention. Conclusion: The introduction of the Lao antimicrobial prescribing guidelines significantly enhanced sixth year medical students’ knowledge of antimicrobial use. Following over one month of guideline exposure and hospital-based training, most students demonstrated improved knowledge to apply appropriate prescribing practices.