Breast cancer is the most commonly diagnosed cancer among women in the Kingdom of Saudi Arabia and other Middle East countries. This analytical cross-sectional study assessed knowledge, attitude towards breast cancer, and barriers to mammogram screening among 414 randomly selected female healthcare workers from multiple healthcare facilities in northern Saudi Arabia. Of the studied population, 48.6% had low knowledge, and 16.1% had a low attitude towards breast cancer risk factors and symptoms. The common barriers to mammogram screening were fear to discover cancer (57.2%) and apprehension regarding radiation exposure (57%). Logistic regression analysis found that lack of awareness regarding mammogram was significantly associated with age (p = 0.030) and healthcare workers category (ref: physicians: p = 0.016). In addition, we found a significant negative correlation between knowledge and barrier scores (Spearman’s rho: −0.315, p < 0.001). It is recommended to develop target-oriented educational programs for the healthcare workers, which would empower them to educate the community regarding the risk factors and the importance of mammogram screening. Furthermore, a prospective study is warranted in other regions of the Kingdom of Saudi Arabia to understand the region-specific training needs for the healthcare workers.