Background Antimicrobial resistance (AMR) is a major global health concern with increasing reports of microorganisms resistant to most of the available antibiotics. There are limited data on antibiotic practices, perceptions and self-medication among Ugandans, necessitating this study. Methods A cross-sectional study was conducted among patients at Kiruddu National Referral Hospital, Kampala, Uganda. A pre-tested interviewer administered a questionnaire that was used to collect data after an informed consent. Chi-square tests and logistic regression were used to assess associations between outcome and exposure variables. A P<0.05 was statistically significant. Results A total of 279 patients (response rate=71%) with a median age of 32 years participated in the study. The majority were females (55.6%, n=155) and from the outpatient department (74.9%, n=209). Overall, 212 (76%) participants had taken an antibiotic in the past 6 months, and some 22.2% (n=47) of the participants had practiced self-medication. Male participants (adjusted odds ratio (aOR)=2.13, 95% confidence intervals (CI): 1.01 to 4.50, P=0.046) and Muslims (aOR=4.37, 96% CI:1.54 to 12.44, P=0.006) were more likely to self-medicate. Employees (aOR=0.06, 95% CI:0.01 to 0.51, P=0.010) and patients with tertiary education (aOR=0.14, 95% CI: 0.02 to 0.81, P=0.028) were less likely to practice self-medication. About 33% (n=70) of the participants had not completed treatment dosage during their last course of antibiotic treatment because of feeling better (60%, n=42), lack of money to purchase the medication (15.7%, n=11) and side effects (10%, n=7). Whereas 169 participants (79.7%) believed that not completing treatment would have an impact on their personal health, only 96 participants (45.3%) believed that this behaviour could affect the health of others. Conclusion Antibiotic misuse is significant among patients in Uganda. Continuous health education programs aimed at informing the public on antimicrobial resistance, and its dangers are recommended to curtail this challenge.