BackgroundEmerging infectious diseases like the Ebola Virus Disease (EVD) pose significant global public health threats. Uganda has experienced multiple EVD outbreaks, the latest occurring in 2022. Frontline healthcare workers (HCWs) are at increased risk, yet there is insufficient evidence of existing knowledge of EVD of these HCWs. We aimed to assess the readiness of Uganda’s emergency HCWs to manage EVD and identify their training needs to inform targeted capacity-building interventions for future outbreaks.MethodsThis multicentre nationwide cross-sectional study was conducted from July to September 2023 among HCWs providing emergency care in 14 secondary and tertiary hospitals across Uganda. Participants were consecutively recruited using the probability-proportional-to-size sampling technique, and data was collected using a self-reported questionnaire. Factors associated with EVD knowledge were identified through a mixed-effect linear model.ResultsData from 691 eligible HCWs with a median age of 32 (IQR: 28–38) was analyzed (response rate: 92%). Only one-third (34.4%, n = 238) had received EVD training in the past year. The median EVD knowledge score was 77.4% (IQR: 71.2 − 83.4%). EVD knowledge was associated with longer professional experience in years (β: 0.21, 95% CI: 0.03 to 0.39, p = 0.024) and higher level of education: diploma (β: 3.37, 95% CI: 1.49 to 5.25, p < 0.001), undergraduate degree (β: 6.45, 95% CI: 4.11 to 8.79) and postgraduate degree (β: 7.13, 95% CI: 4.01 to 10.25, p < 0.001). Being a doctor (β: 2.55, 95% CI: 0.35 to 4.74, p = 0.023), providing care in the obstetrics/gynecology department (β: −1.90, 95% CI: −3.47 to − 0.32, p = 0.018), previous EVD training (β: 2.27, 95% CI: 0.96 to 3.59, p = 0.001) and accessing EVD information through social media (β: 2.52, 95% CI: 1.17 to 3.88, p < 0.001) were also significantly associated with EVD knowledge. HCWs were mostly interested in training on management (92.9%), infection prevention and control (87.1%), and clinical presentation (84.1%), and preferred physical (51.5%) and hybrid (38.2%) training modes.ConclusionOur study shows that the readiness of HCWs in Uganda to respond to EVD outbreaks varies by individual factors and information sources. We recommend targeted training and suggest future research on educational innovations and social media’s potential to fill knowledge gaps.