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.