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Objective Limited data exist on temporal changes in antibiotic use in low and middle-income countries. We evaluated trends in antibiotic use at tertiary hospitals in Uganda.Design Retrospective trend analysis of a repeated point prevalence survey (PPS).Setting and participants This study utilised antibiotic use data from quarterly PPS conducted among inpatients at nine regional referral hospitals in Uganda between October 2020 and December 2023.Outcome measures We determined the proportions of antibiotic use, prescriptions guided by culture and sensitivity tests (CST), WHO AWaRe ( Access, Watch and Reserve) categories, and prescriptions without documented indication. Linear regression was used to derive slope coefficients and 95% confidence interval (CI).Results Of 15,154 patients surveyed, 8,892 (58.7%) received systemic antibiotics. The median age was 23 years (IQR: 11–38), 5,338 (60.5%) were female, and 4,583 (51.5%) were on treatment for infectious syndromes, including sepsis (1,400, 15.7%) and pneumonia (867, 9.8%). The drug utilisation index (DU75) consisted of ceftriaxone, metronidazole, gentamicin and ampicillin, which accounted for 76.9% (12,291/15,989) of total antibiotic use. The distribution of prescribed antibiotics was 46.6% Access, 45.5% Watch, 0.1% Reserve and 7.7% unrecommended combinations. Overall, 5,402 (60.8%) prescriptions were aligned with national guidelines, 2,147 (24.1%) prescriptions were issued without an indication, and CST guided 271 (3%) prescriptions. Over time, there was no significant change in antibiotic prescription prevalence (slope=0.09, CI −0.93 to 1.10) and prescriptions without indication (slope=−0.70, CI −1.79 to 3.98). However, adherence to treatment guidelines (slope=2.06, CI 0.14 to 3.98) and prescriptions based on CST results (slope=0.62, CI 0.12 to 1.13) significantly increased, while ‘ Watch’ antibiotics prescriptions decreased (slope=−0.40, CI −0.63 to –0.17).Conclusions The antibiotic prescription rate remained high, with no significant change over time. Improvements were seen in adherence to treatment guidelines, use of CST and reduced use of ‘ Watch’ antibiotics. Strengthening antibiotic stewardship is recommended to further improve practices.