Purpose Mammography screening (MS) is an underutilized screening tool; although it is provided free of charge to the Saudi community. The present study aimed at assessing knowledge and barriers of mammography screening among women attending primary health centers in Aljouf region, Saudi Arabia. Materials and Methods A cross-sectional study was carried out among 423 women aged 41–75 years attending ten primary health centers in Aljouf region, Saudi Arabia. Structured interviewing questionnaires were used for data collection. SPSS program, version 24 was used for data analysis. Results The least reported risk factors of BC in this study were early menarche (14.9%), first pregnancy after the age of 30 years (18%), and late menopause (18.7%). Concerning knowledge of MS, 50.8% of the women correctly identified that mammogram is the ideal method for detecting BC. The most encountered personal barriers towards MS were lack of information about mammogram (69.5%), fear of exposure to radiation (67.4%), fear of discovery of BC (62.9%), being busy all the time (62.2%), and fear of cancer treatment (61.9%). Regarding economic barriers, 40% of the participants reported that taking sick leave from work is difficult while 37.8% revealed that mammogram is costly. Concerning health system barriers, the most common barriers were fear of error in diagnosis (62.6%), long time to take medical appointment (57%), and preferring not to have a mammogram except after a doctor recommendation (52.7%). The present study showed that women’s education and residence significantly predicted their knowledge. Furthermore, women’s education and income significantly predicted their barriers towards MS. Conclusion Many barriers toward mammography screening were encountered in the present study. Addressing these barriers and raising awareness about MS may be of great value to increase its uptake by Saudi women.