Background Musculoskeletal disorder is a common cause of morbidity, disability, and poor quality-of-life. Its burden among cleaners in developed countries ranges from 56–90%. However, there are a lack of studies in developing countries, particularly in the study area. Thus, the result of this study will help to resolve health problems caused by musculoskeletal disorders among cleaners. Purpose The objective of this study was to identify the prevalence and associated factors of musculoskeletal disorders among cleaners working at Mekelle University. Materials and Methods An institutional-based cross-sectional study was conducted on 270 cleaners. Simple random sampling was used to select the study participants. Data were collected through interview using the standard Nordic Musculoskeletal Questionnaire. Descriptive statistics and bivariate logistic regression were done to identify factors associated with musculoskeletal disorder. In bivariate logistic regression analysis, variables with a P -value<0.25 were modeled to multivariate analysis. Variables with a P -value≤0.05 with 95% confidence interval (CI) in multivariate model were taken as statistically significant. Finally, AOR with 95% CI at a P -value<0.05 was reported. Results This study found that the prevalence of musculoskeletal disorders among cleaners was 52.3% in the past 12 months and 31.8% in the last 7 days. Time pressure (AOR=3.25, 95% CI=1.08–9.77), work experience (AOR=2.49, 95% CI=1.12–5.52), feeling exhausted (AOR=2.68, 95% CI=1.16–6.20), working hours per day (AOR=3.55, 95% CI=1.54–8.20), awkward posture (AOR=15.71, 95% CI=6.47–38.17), and those who work more than 2 hours in sustaining position (AOR=8.05, 95% CI=2.25–28.85) showed a statistically significant association with musculoskeletal disorder. Conclusion Musculoskeletal disorders were commonly reported among cleaners working at Mekelle University. Time pressure, work experience, feeling exhausted, working hours per day, awkward posture, and working >2 hours in sustaining position were statistically significant in their association with musculoskeletal disorders.