Cervico-facial actinomycosis is an infectious, suppurative, and granulomatous disease due to Actinomyces species. Usually, the diagnosis is confirmed by microbiological cultures; however, the need for careful anaerobic handling of specimens often makes it difficult to obtain an effective microbial growth. Therefore, we conducted a retrospective study on biopsy samples from patients with a clinical suspicion of cervico-facial actinomycosis, in order to determine whether accurate histopathological examination could reliably confirm the diagnosis. A retrospective revision of formalin-fixed, paraffin-embedded archival material from 68 cases of cervico-facial lesions, with negative culture for anaerobic/microaerophilic microorganisms, was performed. Twelve serial sections for each case were cut from the paraffin blocks, individually collected on positively charged slides to obtain good section-to-slide adhesion, and stained with hematoxylin and eosin (H&E) and periodic acid-Schiff (PAS). Histopathological examination of the serial sections allowed the identification of bacterial colonies consistent with actinomycetes in 22 cases (32 %). The proposed histopathological examination allowed the retrospective diagnosis of cervical actinomycosis in one-third of clinical specimens that remained misdiagnosed following traditional H&E examination.