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Background Monitoring of microbiological cause of infective endocarditis (IE) remains key in the understanding of IE; however, data from large, unselected cohorts are sparse. We aimed to examine temporal changes, patient characteristics, and in‐hospital and long‐term mortality, according to microbiological cause in patients with IE from 2010 to 2017. Methods and Results Linking Danish nationwide registries, we identified all patients with first‐time IE. In‐hospital and long‐term mortality rates were assessed according to microbiological cause and compared using multivariable adjusted logistic regression analysis and Cox proportional hazard analysis, respectively. A total of 4123 patients were included. Staphylococcus aureus was the most frequent cause (28.1%), followed by Streptococcus species (26.0%), Enterococcus species (15.5%), coagulase‐negative staphylococci (6.2%), and “other microbiological causes” (5.3%). Blood culture–negative IE was registered in 18.9%. The proportion of blood culture–negative IE declined during the study period, whereas no significant changes were seen for any microbiological cause. Patients with Enterococcus species were older and more often had a prosthetic heart valve compared with other causes. For Streptococcus species IE, in‐hospital and long‐term mortality (median follow‐up, 2.3 years) were 11.1% and 58.5%, respectively. Compared with Streptococcus species IE, the following causes were associated with a higher in‐hospital mortality: S aureus IE (odds ratio [OR], 3.48 [95% CI, 2.74–4.42]), Enterococcus species IE (OR, 1.48 [95% CI, 1.11–1.97]), coagulase‐negative staphylococci IE (OR, 1.79 [95% CI, 1.21–2.65]), “other microbiological cause” (OR, 1.47 [95% CI, 0.95–2.27]), and blood culture–negative IE (OR, 1.99 [95% CI, 1.52–2.61]); and the following causes were associated with higher mortality following discharge (median follow‐up, 2.9 years): S aureus IE (hazard ratio [HR], 1.39 [95% CI, 1.19–1.62]), Enterococcus species IE (HR, 1.31 [95% CI, 1.11–1.54]), coagulase‐negative staphylococci IE (HR, 1.07 [95% CI, 0.85–1.36]), “other microbiological cause” (HR, 1.45 [95% CI, 1.13–1.85]), and blood culture–negative IE (HR, 1.05 [95% CI, 0.89–1.25]). Conclusions This nationwide study showed that S aureus was the most frequent microbiological cause of IE, followed by Streptococcus species and Enterococcus species. Patients with S aureus IE had the highest in‐hospital mortality.