Background Healthcare activities restore health and save lives at the same time; however, they can generate hazardous biomedical wastes to a human being or the environment. Generation and disposal of biomedical wastes have become an emerging problem worldwide. Objective To assess knowledge, attitude, and practice about biomedical waste management and associated factors among healthcare professionals in Debre Markos town healthcare facilities, northwest Ethiopia. Methods A cross-sectional study was employed, and data were collected through structured self-administered questionnaire and observational checklist. Data were entered into the Epi-data 3.1 software and exported into SPSS version 20 for analysis. Bivariate and multivariate logistic regression analyses were computed. Variables with a P value of <0.05 in the multivariate logistic regression analysis were considered to explain the presence of statistically significant associations. Result Among 296 healthcare professionals studied, 168 (56.8%), 196 (66.2%), and 229 (77.4%) had adequate knowledge, favorable attitude, and adequate practice score, respectively. Regarding associated factors, MSc and MD+ (AOR: 4, 95% CI: (1.37, 149.52)), BSc holders (AOR: 2.53, 95% CI: (1.47, 4.38)), and availability of color-coded bins (AOR: 7.68, 95% CI: (3.30, 17.89)) were identified more likely to contribute for adequate knowledge, favorable attitude, and adequate practice scores, respectively. Conclusion The level of knowledge, attitude, and practice scores were not satisfactory. Majority of the healthcare professionals did not access biomedical waste management training. Educational level, use of visual aid, and availability of color-coded bins in the department were identified as a factor for biomedical waste management. Regular training should be given to healthcare professionals.