The aim of this study, performed between September to November 2010, was to provide insight into Italian girls' and women's knowledge of sexual health and sexuality, in particular regarding the physiology of human reproduction, contraception, sexuality, cervical cancer screening and abortion. The data used were obtained through face-to-face interviews with two groups of women, performed by a trained interviewer using a structured multiple-choice questionnaire. The first group was composed of young women aged 17 to 20 years, randomly selected from five high-school institutes in the inner-city area of Perugia (Italy), while the second group was made up of women aged 30 to 43 years, recruited amongst women undergoing postpartum checks at a regional hospital. The number of correct answers for each group of questions and the total number of correct answers in the questionnaire were calculated. A Poisson regression model was used to identify the main determinants of answering correctly. Adult women gave a higher rate of correct answers for each topic, except for those related to abortion, contraception, and HPV. Characteristics of adult women associated with a better knowledge about sexuality were: discussing about sexuality with friends or partner, being Italy-born and having an open and relaxed attitude toward sex. For younger women, better knowledge was associated with being Italy-born, having attended affectivity education courses and having an open and relaxed attitude towards sex. Results suggest that no sex education or information source by itself is effective but what is needed is a combination of various information sources, both formal and informal, including parent-adolescent communication.