This case series included a tomographic, microbiological, and histopathological description of 15 secondary apical periodontitis (SAP) lesions obtained by apical microsurgery performed in 10 patients to better understand the etiology and pathogenesis of SAP. Preoperative tomographic analyses were performed through Cone beam computerized tomography - Periapical index (CBCT-PAI), and apical microsurgeries were then carried out. The removed apices were used for microbial culturing and for molecular identification using PCR for the detection of 5 strict anaerobic bacteria (P. gingivalis, P. intermedia, P. nigrescens, T. forsythia, and T.denticola) and 3 viruses Herpes simplex viruses (HSV), Cytomegalovirus (CMG) and Epstein-Barr Virus (EBV) by nested PCR. The removed apical lesions were histologically described. Univariate statistical analyses were performed by using STATA MP/16 (StataCorp LLC, College Station, TX, United States). CBCT-PAI analyses revealed PAI 4 and PAI 5 score lesions that involved cortical plate destruction. Eight SAPs were positive by culture, while nine SAP lesions were positive by PCR. Fusobacterium species were the most frequently cultured organisms in 7 SAP lesions, followed by D. pneumosintes in 3. In contrast, by single PCR, T. forsythia and P. nigrescens were detected in 5 lesions, T. denticola in 4 lesions, and P. gingivalis in 2 lesions. Twelve periapical lesions were granulomas, and the remaining three SAP lesions were radicular cysts. In conclusion, this case series study revealed that secondary apical lesions presented tomographic involvement of PAI 3 to 5, and that most SAP lesions were apical granulomas containing anaerobic and facultative microorganisms.