Antibiotic resistance (ABR) is a major public health issue, associated with increased patient morbidity and mortality globally, with significantly higher rates in low- and middle-income countries (LMICs). Assessment of contextual factors, such as information, education, infrastructure and regulations are important for developing local solutions against ABR. To determine the knowledge and practices of healthcare workers (HCWs) towards ABR in hospitals in Sudan. A survey was conducted in three different hospitals in Khartoum, Sudan from February to December 2020. HCWs of different specialties and expertise were invited to participate. Data were descriptively analysed using Statistical Package for Social Sciences (SPSS). ABR was identified as a big challenge by 89% of 345 HCWs who participated. The results show that 79% of doctors don't rely on the clinical microbiology laboratory (CML) results for antibiotic prescription or clinical decision-making. Sixty percent of HCWs agreed there are infection prevention and control (IPC) guidelines in their hospital, but 74% of them don't have access to them, and infrequently receive relevant IPC training. Furthermore, HCWs obtain ABR information from other colleagues informally, not through local data or reports. Despite adequate knowledge of ABR locally, there are significant contextual technical challenges facing HCWs in Sudan, such as availability of policies and accurate data from CMLs. The results indicate a poor link between HCWs and the CMLs for infection management and it is essential to improve communication between the different hospital departments with regard to ABR transmission, and ensure the effectiveness of local IPC policies based on locally available data.