Background: Adolescence is an important period of physical, emotional and sexual development during which young people may become vulnerable to sexually transmitted infections and unintended pregnancy. Adequate knowledge of safe sex strategies is important; however, knowledge may not necessarily translate into appropriate sexual health practices. Objective: To assess the knowledge and practice of safe sex strategies and identify factors influencing sexual health practices among senior secondary school students at University of Benin Demonstration Secondary School, Benin City, Nigeria. Methods: This was a cross-sectional study involving 243 senior secondary school students. A stratified random sampling technique was used to select participants from SS1 to SS3. Data were collected using a structured, self-administered questionnaire assessing sociodemographic characteristics, knowledge of safe sex strategies, sexual practices and factors influencing safe sex practices. The instrument underwent face and content validation and had a Cronbach’s alpha coefficient of 0.75 following pilot testing. Data were analysed using SPSS version 26 and summarised using descriptive statistics. Results: Of the 243 respondents, 139 (57.2%) were female and 126 (51.9%) were aged 14–16 years. Overall, 201 (82.9%) respondents had good knowledge of safe sex strategies, while 124 (51.0%) had good safe sex practices. One hundred and sixty (65.8%) had attended a class on safe sex education, while 159 (65.4%) had previously heard the term “safe sex.” Sixty-nine (28.4%) respondents reported having had sexual intercourse, 149 (61.3%) reported practising abstinence, and only 60 (24.7%) had ever undergone testing for a sexually transmitted infection. Only 112 (46.1%) had discussed sexual health with a parent, teacher or health professional. Conclusion: Knowledge of safe sex strategies was high among the students, but this was not matched by a similarly high level of safe sex practice. Strengthening comprehensive school-based sexuality education, improving parent-adolescent communication and increasing access to confidential youth-friendly sexual and reproductive health services may help bridge the gap between knowledge and practice.