A variety of methods, including direct examination of stained smears, antigen detection, routine and special cultures, and histopathologic evaluation are available for investigation of head and neck infections. Newer rapid molecular techniques with increased sensitivity and specificity are becoming available at many centers. Evaluation of specific causes in head and neck infections is complicated by the tendency for polymicrobial infections, difficulty in obtaining adequate specimens, and the presence of a diverse endogenous microflora at various mucosal sites that also can include true pathogens. These aspects of laboratory investigation for head and neck infections are reviewed in this article.