Background Breast cancer contributed to about 2.3 million cancer cases among women globally and 685,000 women died from it in 2020. Breast self-examination is considered one of the many breast cancer-screening techniques in Low and Middle Income Countries. It is a useful tool if practiced every month by women above 20 years of age and combined with other screening methods such as mammography, Breast Clinical Examination, breast ultrasound and Magnetic Resonance Imaging. However, the practice of BSE is poor in many developing countries. Therefore, this research is intended to measure the level of BSE practice and determine the associated factors among female healthcare professionals in four district hospitals in Kigali, Rwanda. Materials and Method A cross-sectional study was conducted among 221 randomly selected female healthcare professionals in four district hospitals in Kigali, Rwanda. A self-administered structured questionnaire was used as data collection instrument. Sample statistics such as frequency, proportions and mean were used to recapitulate the findings in univariate analysis. Multiple logistic regression analysis was employed to identify statistically significant explanatory variables that predict the outcome variable, breast self-examination practice, based on an adjusted odds ratio, 95% confidence level and p-value < 0.05. Results Breast self-examination was practiced by 43.53% of female healthcare professionals. Attitude towards BSE and BC was the only predictor variable that is significantly associated with BSE practice [AOR=1.032; 95% CI (1.001, 1.065), p-value=0.042]. Knowledge on BSE and BC, age, having family history of breast cancer were not significantly associated with BSE practice in the multi-variate analysis. Conclusion The BSE practice was found to be low. Attitude towards breast cancer and BSE was positively associated with BSE practice. This suggests the need for continuous medical education on BSE and BC to increase the knowledge level of female healthcare professionals and enhance the attitude of female healthcare professionals in order to increase their BSE practice. This could in turn increase the knowledge and practice of BSE among the general public.