Abstract Aim Fatality of infective endocarditis (IE) is high worldwide, and its diagnosis remains a challenge. The objective of the present study was to compare the clinical characteristics and outcomes of patients with culture-positive (CPIE) vs. culture-negative IE (CNIE). Methods and results This was an ancillary analysis of the ESC-EORP EURO-ENDO registry. Overall, 3113 patients who were diagnosed with IE during the study period were included in the present study. Of these, 2590 (83.2%) had CPIE, whereas 523 (16.8%) had CNIE. As many as 1488 (48.1%) patients underwent cardiac surgery during the index hospitalization, 1259 (48.8%) with CPIE and 229 (44.5%) with CNIE. The CNIE was a predictor of 1-year mortality [hazard ratio (HR) 1.28, 95% confidence interval (CI) 1.04–1.56], whereas surgery was significantly associated with survival (HR 0.49, 95% CI 0.41–0.58). The 1-year mortality was significantly higher in CNIE than CPIE patients in the medical subgroup, but it was not significantly different in CNIE vs. CPIE patients who underwent surgery. Conclusion The present analysis of the EURO-ENDO registry confirms a higher long-term mortality in patients with CNIE compared with patients with CPIE. This difference was present in patients receiving medical therapy alone and not in those who underwent surgery, with surgery being associated with reduced mortality. Additional efforts are required both to improve the aetiological diagnosis of IE and identify CNIE cases early before progressive disease potentially contraindicates surgery.