Endomyocardial biopsy (EMB) is an invasive procedure originally developed for the monitoring of heart transplant rejection. Over the year, this procedure has gained a fundamental complementary role in the diagnostic work-up of several cardiac disorders, including cardiomyopathies, myocarditis, drug-related cardiotoxicity, amyloidosis, other infiltrative and storage disorders, and cardiac tumours. Major advances in EMB equipment and techniques for histological analysis have significantly improved diagnostic accuracy of EMB. In recent years, advanced imaging modalities such as echocardiography with three-dimensional and myocardial strain analysis, cardiac magnetic resonance and bone scintigraphy have transformed the non-invasive approach to diagnosis and prognostic stratification of several cardiac diseases. Therefore, it emerges the need to re-define the current role of EMB for diagnostic work-up and management of cardiovascular diseases. The aim of this review is to summarize current knowledge on EMB in light of the most recent evidences and to discuss current indications, including challenging scenarios encountered in clinical practice.