Breast cancer is the second most common cancer among females worldwide and can often be detected at an early stage through breast self-examination (BSE). However, in many developing countries, most cases are diagnosed at advanced stages. This study aimed to assess the knowledge and practice of BSE among women of reproductive age in Saudi Arabia. This study adopted a community-based descriptive-analytical cross-sectional design. A stratified simple random sampling technique was used, with 50 participants from each region. Almost 500 Saudi women who fulfilled the inclusion criteria were enrolled in this study, and of these, 32.8% were within the age group of 20–29, 30.4% were within the age group of 30–39, and 32.8% were within the age group of more than 40 years. The overall knowledge score regarding BSE items among participants was 38%. Significant associations were found between breast cancer knowledge scores and demographic factors such as age group, educational level, marital status, region, and residence area (P < 0.05). Regarding knowledge of breast cancer risk factors, 48% of participants demonstrated poor knowledge, 43% had moderate knowledge, and only 9% had good knowledge. Effective prevention of breast cancer requires awareness and understanding of its risk factors. It is important for young women, starting from puberty, to be educated about potential changes in breast tissue and to receive proper training in BSE techniques from healthcare professionals.