Background & aim: Sexual health education is one of the responsibilities of healthcare workers at schools, which can reduce the risk of sexually transmitted diseases such as AIDS, unwanted pregnancy, abortion, substance abuse, sexual violence, and suicidal tendencies. This study aimed to investigate healthcare workers’ competence in sexual health education for female adolescents at schools. Methods:This cross-sectional study was conducted on 300 healthcare workers, responsible for sexual health education at schools in 2015. A valid and reliable researcher-made questionnaire was completed by the healthcare workers in order to assess their competence in sexual health education at healthcare centers of Khuzestan, Iran. To assess the competence of the participants (i.e., knowledge, attitude, confidence, and performance), descriptive statistics were calculated for quantitative variables. Also, mean, standard deviation, frequency, and percentage were calculated for qualitative variables. Pearson’s correlation test was performed to assess the relationship between the subjects’ knowledge, attitude, confidence, and performance. Also, the association between demographic variables and participants’ knowledge, attitude, confidence, and performance was evaluated, using analysis of variance (ANOVA). Data were analyzed, using SPSS version 21.0. Results: Knowledge, attitude, and confidence of healthcare workers in sexual health education were desirable. However, the subjects showed a poor performance in teaching students the required skills to control their emotions, instincts, homosexual tendencies, and masturbation. There was a significant correlation between performance, attitude, and confidence, knowledge and attitude, performance and confidence, and confidence, performance, and attitude (P<0.05). Academic field and educational level had significant impacts on knowledge; also, employment status influenced the subjects’ knowledge and attitude. However, statistical analysis did not indicate a significant association between other variables (P<0.05). Overall, the knowledge, attitude, confidence, and performance of the midwifery staff were more desirable than other healthcare providers. Conclusion: Despite the adequate knowledge, positive attitude, and confidence of healthcare workers, their performance on sexual health education, especially taboo topics, was unacceptable. To eradicate the adolescents’ problems in sexual health, it is necessary to conduct broad investigations to identify the underlying causes of healthcare workers’ weak performance in this context.