Breast cancer (BC) is the most prevalent cancer among women, and it typically presents late in developing countries like the Democratic Republic of the Congo (DRC), leading to higher mortality rates. Late detection at advanced stages of breast cancer can be attributed to the absence of appropriate screening programs and low levels of awareness. To evaluate the level of BC knowledge among healthcare workers (HCWs) and identify determinants of good BC knowledge. An analytical cross-sectional survey was conducted from March 1 to 31, 2022 involving HCWs practicing in Kinshasa, DRC. Data were collected using a questionnaire administered through direct interviews. Bivariate and multivariate regression techniques were applied. The study interviewed 543 HCWs, with a median age of 35 years (interquartile range: 29-43). Of these, 61% had good BC knowledge, while 39% had poor BC knowledge. Multivariate analysis revealed that HCWs aged 50 years and over (adjusted odds ratio [aOR] = 2.3 [1.2-4.5]), female HCWs (aOR = 1.8 [1.1-2.4]), HCWs working in public healthcare facilities (aOR = 1.5 [1.1-2.5]), and HCWs who had received training on BC (aOR = 1.9; 95% CI: 1.5-3.3) were determinants of good BC knowledge. This study found that 61% of the surveyed HCWs had good BC knowledge. However, there is still room for improvement in terms of knowledge dissemination. Therefore, it is important to implement continuing medical education programs that focus on raising awareness and improving BC knowledge among HCWs.