Antibiotic resistance is a major emerging global public health threat. Farmers in the Khartoum state are believed to misuse antibiotics in animal farming leading to daily exposure to resistant bacteria and antibiotic residues. Hence, farmers are at potential risk exposure to bacteria, zoonotic infection and toxicity. We hypothesized that farmers’ misuse of antibiotics could be due to their ignorance of the importance of optimal use of antibiotics, the potential health hazards and the economical waste associated with antibiotic misuse practices. In the present study, we investigated knowledge and practices among farmers regarding antibiotic use and resistance. For this purpose, a cross-sectional study was conducted in Khartoum state where data were collected from 81 farmers using structured interviews. Data were analysed both quantitatively and qualitatively. Fifty-two per cent of farmers were uneducated or had studied for <6 years. The majority reported antibiotic use for treatment and prevention while only 5% stated use for growth promotion. Antibiotic group treatment for both sick and healthy animals was commonly practiced among most farmers. The most commonly used group of antibiotics was the quinolones, which was reported by one-third. Only 30% of the farmers had heard of antibiotic resistance and provided their definition. Almost half were not aware of the commonly transferred zoonotic infections between humans and animals. The farmers consume 1–2 meals/day from their own farm products. A significant association between low education, poor knowledge of farmers on antibiotic use, antibiotic resistance and zoonotic infections was found. This association may play a vital role in the present practiced misuse of antibiotics. Our findings on farmers’ practices could be used as baseline information in defining the gaps related to antibiotic use and resistance in animal farming in Sudan. It can thus serve as a foundation for future interventions.