Food safety knowledge, attitudes and practices among hospital food service staff are crucial in the prevention of foodborne disease outbreaks, as hospitalized patients are more vulnerable to potential hazards. This study, therefore, sought to assess the food safety knowledge, attitudes and practices of food service staff in Bangladeshi hospitals. A cross-sectional study was conducted among 191 food service staff from seven different hospitals in Dhaka and Chattogram from October 2021 to March 2022 using pretested questionnaires. Multiple linear regression was used to identify the factors associated with the food safety knowledge, attitudes and practices. The findings showed moderate knowledge but high levels of attitudes and practices of food safety among hospital food handlers. Food safety knowledge was significantly higher among males, participants from private hospitals and participants working in a hospital that had a food service supervisor and dietitian in charge of food service operations. Moreover, participants from private hospitals and participants working in a hospital that had a food service supervisor and dietitian in charge of food service operations had more positive attitudes and better practices regarding food safety. Hospital management should consider these factors for enhancing food handlers’ knowledge and increase training and supervision on food safety practices to reduce foodborne diseases and outbreaks.