OBJECTIVE The purpose of the study is to evaluate and compare the biological and chemical-physical properties of four different root-end filling materials. METHODS Cytotoxicity towards murine odontoblasts cells (MDPC-23) was evaluated using the Transwell insert methodology by Alamar blue test. Streptococcus salivarius, S. sanguis, and S. mutans strains were selected to evaluate the antimicrobial activity by agar disc diffusion test. Solubility was determined after 24 h and 2 months. pH values were measured after 3 and 24 h. To evaluate radiopacity, all materials were scanned on a GE Healthcare Lunar Prodigy. RESULTS Excellent percentage of vitality were obtained by mineral trioxide aggregate (MTA)-based materials and Biodentine. MTA-Angelus, ProRoot MTA, and Intermediate Restorative Material (IRM) showed the highest values for the inhibition zones when tested for S. mutans, while Biodentine showed the largest inhibition zone when tested for S. sanguis. All the materials fulfilled the requirements of the International Standard 6876, demonstrating low solubility with a weight loss of less than 3%. No significant reduction in pH value was demonstrated after 24 h. ProRoot MTA and MTA-Angelus showed the highest values of radiographic density. CONCLUSIONS The differences showed by the root-end filling materials tested do not cover completely the ideal clinical requests.