This study assessed the prevalence and microbial interactions of Fusobacterium nucleatum and Fusobacterium necrophorum in primary endodontic infections from a Brazilian population and their antimicrobial susceptibility to some antibiotics by the E-test. One hundred ten samples from infected teeth with periapical pathologies were analyzed by culture methods. Five hundred eighty individual strains were isolated; 81.4% were strict anaerobes. F. nucleatum was found in 38 root canals and was associated with Porphyromonas gingivalis, Prevotella spp., and Eubacterium spp. F. necrophorum was found in 20 root canals and was associated with Peptostreptococcus prevotii. The simultaneous presence of F. nucleatum and F. necrophorum was not related to endodontic symptoms (p > 0.05). They were 100% susceptible to amoxicillin, amoxicillin/clavulanate, and cephaclor. Fusobacterium spp. is frequently isolated from primary-infected root canals of teeth with periapical pathologies. Amoxicillin is a useful antibiotic against F. nucleatum and F. necrophorum in endodontic infections and has been prescribed as the first choice in Brazil.