Butcheries are the leading retailers of beef in Uganda and their level of compliance with food safety standards is unknown. The aim of this study was to determine the compliance by beef vendors in Kamuli district with the US 736:2019 standard for hygienic requirements for butcheries. A survey questionnaire and observation checklist on sanitation, hygiene, and beef handling were used to collect data from 60 butcheries. More than 75% of beef vendors complied with the inspection, storage, and some sanitation and hygiene requirements, however, many violated the transportation and construction requirements. Self-reported surveys revealed that 96.7% of vendors sold inspected beef, 83.3% of butchery facilities were inspected at least once a month and all vendors stored beef for less than 36 hours. Beef vendors (76.7%) reported washing beef handling tools with water and soap whereas 96.7% cleaned butcheries every day. Sixty per cent (60%) of vendors transported beef using motorcycles, and 23.3% used tricycles. All beef vendors observed had short hair, short fingernails, and did not wear jewellry. Only 15% of vendors wore protective clothing when handling beef. Butcheries had wooden walls (71.7%), and their floors were either wooden or bare ground (65%). This study demonstrated a need for food safety interventions to emphasize sanitation and personal hygiene practices, safe transportation of beef, and the hygienic construction of butchery facilities.