Background Meat is rich in essential proteins and valuable nutrients for human health. Despite these benefits, it is a favorable medium for microbial growth and transmission to humans unless recommended safety procedures are followed. This research aimed to assess the level of knowledge and practice of the abattoir and butcher shop workers who were working in the meat value chain. Methods The cross-sectional study design, using structured questionnaire interviews was used to assess the knowledge and practice of abattoir and butcher shop workers. A total of 226 randomly selected workers were participated in this study and bivariate logistic regression was used for data analysis. Results Of 226 total participants, 46% were abattoir workers and 54% were butcher shop workers. Majority (88.9%) of the participants did not know about meat safety and 74.3% of the participants had insufficient practices. The cumulative knowledge of both abattoir and butcher shop workers was significantly associated (p = 0.001) with their knowledge of the presence of diseases causing agents in meat, the source of meat contamination, and the common critical points of meat contamination along the meat value chain. The cumulative practice of abattoir workers was significantly associated with their practices on daily meat transporting vehicle washing (p = 0.007), reducing meat contamination (p = 0.001), duration of animal fasting before slaughter (p = 0.039), cleaning of the animal body before slaughter (p = 0.002), cleaning material used in the abattoir (p = 0.003), disposal of abattoir waste (p = 0.002), and type of biosecurity measures used (p = 0.013). Similarly, the cumulative practice of butcher shop workers was associated (p = 0.001) with their practices of attracting customers, storing remaining meat from daily sales, and measures on contaminated food. Employment of the participants was significantly associated with both the cumulative knowledge (p = 0.007) and practice (p = 0.001) of the participants while the age of the participants was associated (p = 0.001) with only their cumulative practices. Conclusions In general, the participants’ food safety knowledge and practice were unsatisfactory. As a result, the integrated food safety policy formulation in a One Health framework, and collaborative awareness creation among different food safety stakeholders were recommended.