Antimicrobial stewardship (AMS), as one of the global strategies to promote responsible use of antimicrobials to prevent antimicrobial resistance (AMR), remains poor in many low-and middle-income countries (LMICs). We implemented a project aimed at strengthening AMS in Wakiso district, Uganda using a One Health approach. A total of 86 health practitioners (HPs), including animal health workers, and 227 community health workers (CHWs) participated in training workshops, and over 300 pupils from primary schools were sensitized on AMR, AMS, and infection prevention and control (IPC). We further established two multidisciplinary online communities of practice (CoPs) for health professionals and students, with a current membership of 321 and 162, respectively. In addition, a Medicine and Therapeutics Committee (MTC) was set up at Entebbe Regional Referral Hospital. The project evaluation, conducted three months after training, revealed that the majority of the HPs (92.2%) and CHWs (90.3%) reported enhanced practices, including improved hand washing (57.3% and 81.0%, respectively). In addition, 51.5% of the HPs reported a reduction in the quantity of unnecessary antibiotics given per patient. This project demonstrates that AMS interventions using a One Health approach can promote understanding of the prudent use of antimicrobials and improve practices at health facilities and in communities.