Bacteria play a major role in the etiology of apical periodontitis. Traditionally it has been held the microorganisms are present in necrotic tissue in the root canal system and in tubules of the root dentin whereas the periapical tissues are free of bacteria. However, it is reported the presence of bacterial in the periapical lesions. They may form the biofilm and survive in the periapical tissues. Especially high incidence of biofilm is reported in the refractory periapical lesions. treatment was presented in the left maxillary first premolar with a long-standing fistula and apical calculus. Also. the role of biofilm and its treatment were discussed.