Pre-marital medical examination is a key preventive public health strategy aimed at reducing hereditary and infectious diseases and supporting healthy family formation. Despite its importance, data from Libya - particularly among university students - remains limited. This study assessed the level of awareness, knowledge, and acceptance of pre-marital medical examination among college and institute students in Nalut City, Libya. It examined the association between educational/cultural level and awareness of pre-marital medical examination. A descriptive cross-sectional study was conducted among 600 students aged 19-27 years from colleges and higher institutes in Nalut City, Libya, in 2025. Data were collected using a self-designed electronic structured questionnaire covering sociodemographic characteristics, acceptance of pre-marital medical examination, knowledge of included tests, and attitudes toward the awareness program. Data were analyzed using SPSS version 25, and associations were tested using the Chi‑square test. Most of the participants accepted pre-marital medical examinations and acknowledged their importance in preventing marital and health problems (95.8%). A significant association was observed between educational/cultural level and awareness of the necessity of pre-marital medical examination (p=0.007). Although general awareness was high, knowledge of specific hereditary conditions such as thalassemia was comparatively limited. Libyan university students demonstrate high acceptance of pre-marital medical examination, but insufficient detailed knowledge of specific genetic disorders. Strengthening targeted educational programs within universities is essential to improve comprehensive understanding and promote informed health decisions.