BACKGROUND: Use of antibiotics to treat humans and animals is increasing worldwide, but evidence from low- and middle-income countries (LMICs) is limited. We conducted cross-sectional surveys in households and farms in Uganda to assess patterns of antibiotic use among humans and animals. METHODS: Between May and December 2018, a convenience sample of 100 households in Nagongera (rural), 174 households in Namuwongo (urban) and 115 poultry and piggery farms in Wakiso (peri-urban) were selected and enrolled. Using the 'drug bag' method, participants identified antibiotics they used frequently and the sources of these medicines. Prevalence outcomes were compared between different sites using prevalence ratios (PRs) and chi-squared tests. RESULTS: Nearly all respondents in Nagongera and Namuwongo reported using antibiotics to treat household members, most within the past month (74.7% Nagongera versus 68.8% Namuwongo, P = 0.33). Use of metronidazole was significantly more common in Namuwongo than in Nagongera (73.6% versus 40.0%, PR 0.54, 95% CI: 0.42-0.70, P < 0.001), while the opposite was true for amoxicillin (33.3% versus 58.0%, PR 1.74, 95% CI: 1.33-2.28, P < 0.001).Veterinary use of antibiotics within the past month was much higher in Wakiso than in Nagongera (71.3% versus 15.0%, P < 0.001). At both sites, oxytetracycline hydrochloride was the most frequently used veterinary antibiotic, but it was used more commonly in Wakiso than in Nagongera (76.5% versus 31.0%, PR 0.41, 95% CI: 0.30-0.55, P < 0.001). CONCLUSIONS: Antibiotics are used differently across Uganda. Further research is needed to understand why antibiotics are relied upon in different ways in different contexts. Efforts to optimize antibiotic use should be tailored to specific settings.