Foodborne zoonosis is a longstanding global issue that limits and continues to threaten the food production industry and public health in several countries. The study’s objective was to evaluate the dairy farmers’ knowledge, attitudes, and practices about milk-borne pathogens in the Eastern Cape province, South Africa. A total of 139 dairy farmers were interviewed using a semi-structured online questionnaire. The pathogens of interest were Brucella spp., Escherichia coli, Listeria monocytogenes, Salmonella spp., Staphylococcus aureus, and Cryptosporidium. Only 20.9% of dairy farmers reported knowledge of Brucella spp. as a milk-borne pathogen. The most known pathogen was E. coli (54.7%), followed by Listeria spp. (41.0%), Staphylococcus spp. (38.8%), and Salmonella spp. (35.3%). In this study, knowledge of milk-borne pathogens was statistically associated (p<0.05) with workplace position. Only a few participants (37.2%) showed knowledge of abortion as an important clinical sign of foodborne pathogens. Also, 84.1% of dairy farmers indicated that they consume unpasteurized milk and sour milk (77%). Some respondents (18.0%) do not believe assisting a cow during calving difficulty without wearing gloves is a risk factor for zoonosis. Knowledge assessment is essential in developing countries that have experienced a foodborne outbreak, such as South Africa. There is an urgent need to educate dairy farmers about milk-borne zoonosis to minimize the threat to food security and public health.