the present study aimed to assess the knowledge and attitudes of medical and nursing students at the University of Patras, western Greece, regarding sexually transmitted infections (STIs), sexual behavior and STI prevention measures, as well as the level of future healthcare professionals' education. A descriptive, cross-sectional study was conducted. A total of 231 medical and nursing students (n = 106 medical, and n = 125 nursing) completed and returned the pre-tested study questionnaire. Most participants (77.5%) were females and46.1% were in the age group of 18-21 years. Syphilis, HIV/AIDS, and Hepatitis B were regarded as STIs by 65.8% of them. Medical students could predominantly list the widely known STIs compared to nursing students (p = 0.004). Regarding HIV/AIDS, 72.7% of the respondents reported that it is transmitted sexually and through blood transfusion. However, medical students were better informed than nursing students (p = 0.001). Medical students as well as students in the final year of their studies were found to be better informed about the vaccines available to prevent STIs. Regarding the question about what constitutes a risky sexual behavior, 71.4% answered sexual intercourse without the use of condom and 18.6% indicated having sex with an unknown partner. Most participants (69.7%) were satisfied with the education provided by their institution and no statistically significant difference was observed between medical and nursing students. Almost all students (97.8%) agreed that the course/subject of sex education must be included in school programs. A comprehensive analysis of knowledge and attitudes of Greek medical and nursing students regarding STIs, prevention measures and education level was conducted. The results of the present study could assist in the development of targeted training courses that can improve healthcare professionals' knowledge and ability to manage STIs.