The engagement of a large number of people in big-scale cooking raises the danger of food contamination due to incorrect handling, whether deliberate or unintentional. Contamination during large-scale production poses a serious hazard to consumer health and has significant financial implications for a nation. This study aimed to investigate the food safety knowledge and practices of institutional food handlers in Bangladesh, considering the growing concern surrounding this issue and the lack of available information on foodborne illnesses related to institutions. In addition, the study aimed to determine the factors influencing both knowledge and practices. A cross-sectional study was conducted from June to September 2022, involving 408 institutional food handlers. The sample size was determined using Cochran's formula, and data was collected through purposive sampling. The participants were interviewed in person and completed a pilot-tested questionnaire. A multiple linear regression analysis was conducted to determine the factors related to food safety knowledge and practices. The majority of participants were female (71.3%) and aged between 26 and 35 (mean age 34.53 ± 9.06 years). They were most knowledgeable about hand hygiene and food separation but lacked knowledge about foodborne pathogens and food storage. Thawing food at room temperature was the most inappropriate practice (86%). The mean scores for knowledge and practice were found to be 16.11 ± 2.76 on a 26-point scale (61%), and 9.59 ± 2.07 on a 15-point scale (64%), respectively. Rural food handlers, those with higher education, working more than 10 h per day, and being familiar with HACCP, had higher knowledge. Food handlers aged 18 to 25, with higher income, working in private institutions, having food safety authority knowledge, actively engaging in food safety training, working more than 10 h per day, and having a positive health perception, had better food safety practices.The results of this study reinforce the notion that institutional food handlers would benefit from enhanced exposure to food safety interventions, active participation in training sessions, and strict adherence to food hygiene regulations in their food handling knowledge and practices.