Aims After the introduction of the European Society of Cardiology (ESC) guidelines on prevention, diagnosis, and treatment of infective endocarditis (IE) in 2009, prophylaxis for patients at risk became less strict. We hypothesize that there will be a rise in IE after the introduction of the guideline update. Methods and Results We performed a nationwide retrospective trend study using segmented regression analysis of the interrupted time series. The patient data were obtained via the national healthcare insurance database, which collects all the diagnoses nationwide. We compared the data before and after the introduction of the 2009 ESC guideline. Between 2005 and 2011, a total of 5213 patients were hospitalized with IE in the Netherlands. During this period, there was a significant increase in IE from 30.2 new cases per 1 000 000 in 2005 to 62.9 cases per 1 000 000 in 2011 (P < 0.001). In 2009, the incidence of IE increased significantly above the projected historical trend (rate ratio: 1.327, 95% CI: 1.205–1.462; P < 0.001). This coincides with the introduction of the 2009 ESC guideline. After the introduction of the ESC guideline, the streptococci-positive cultures increased significantly in the following years 2010–11 from 31.1 to 53.2% (P = 0.0031). Conclusion This observational study shows that there has been a steady increase in the IE incidence between 2005 and 2011. After the introduction of the 2009 ESC guidelines, the incidence increased more than expected from previous historical trends. Furthermore, there was a significant increase in streptococci-related IE cases.