Background Meat is subjected to contamination from a variety of sources during slaughter and sale. Globally, an estimated 600 million people per year are affected by illness from poor meat handling, of which 420,000 result in deaths. However, evidence on the magnitude and factors associated with hygienic practice in low-income countries limits the extent of this underestimation of the problem. The aim of this study was to assess the magnitude of meat hygiene practices and factors associated with poor practices among food handlers in Gambela, Ethiopia. Methods This institution-based cross-sectional study was conducted in Gambela town. Systematic sampling at random was used to select workers from butchers’ houses, meat-selling shops, and restaurants in Gambela. A sampling frame of 1,080 workers was obtained from the total number of restaurants, meat-selling shops, and restaurants in the town. A sampling interval (K) was calculated. Finally, a participant was selected for every two individuals until the final 422 individuals were sampled. Data were collected using structured questions through interviews and observations. Data collectors were trained and oriented on the questionnaires before data collection, and they were supervised throughout the data collection period. Results This study showed a low prevalence of good hygiene practices of 40.6% (36.0–45.6) among meat handlers at butcher houses and restaurants in Gambela, southwest Ethiopia. Low levels of good hygiene practices were significantly related to education (i.e., having a secondary education) (AOR: 95% C.I., 2.51: 1.17–5.41) and food hygiene training (i.e., receiving training on general safe food hygiene) (AOR: 95% C.I., 5.11: 2.55–10.23). Conclusion This study finds evidence of a low level of hygiene practices among meat handlers at butcher houses and restaurants in Gambela, Ethiopia. Low hygiene practices were significantly associated with the education level of food handlers and meat hygiene training. Intensive food safety training and providing a comfortable work environment will help them attract educated workers.