Motivation is the level of a person's willingness to put forth and maintain an effort in support of organizational goals. However, motivation towards task execution is affected by the organization and individual goals. For instance, low morale among the staff can damage the quality of service delivery. Hence, this study was intended to assess the working motivation status and factors associated with it among health professionals at Debre Markos Comprehensive Specialized Hospital. A hospital-based cross-sectional study was employed. Stratified sampling techniques were used to extract sample from each job category proportionally. To make the distribution fair, all health workers were grouped according to their job title and selected by using the lottery method from each group. A standardized, self-administered questionnaire was used to collect data. Data was checked, coded, and entered into EpiData 3.1 and exported for analysis into SPSS 25. Variable in the multivariable logistic regression model with a p value of < 0.05 at 95% CI were taken as significantly associated to motivation status. A total of 319 people were involved, with a 100% response rate. 20.4% of health professionals were motivated at Debre Markos Comprehensive Specialized Hospital. Job satisfaction (AOR 6.46, 95% CI 1.72, 24.35), the presence of adequate medical supplies (AOR 5.01, 95% CI 1.23, 25.37), work place security (AOR 6.78, 95% CI 1.498, 30.72), and the presence of training opportunities in health facilities (AOR 2.23, 95% CI 1.01, 4.96) were significant factors associated with motivation status. The proportion of motivated health professionals was very low compared to previous studies in Ethiopia. The presence of security at work, adequate medical equipment, drugs, and supplies, job satisfaction, and the presence of training opportunities were predominant motivational factors. The hospital administration needs to give priority and work to safeguard security, ensure adequate medical supplies, and offer training to improve their satisfaction and motivation.