Immunohistochemistry is commonly used to detect and characterize infectious agents in diagnostic pathology. The principal advantage of immunohistochemistry over other antigen detection techniques is the ability to identify antigen within the context of histologic lesions. Although epitope masking attributed to formalin fixation, especially prolonged fixation, has been considered a limiting factor in diagnostic immunohistochemistry, only a few studies have evaluated the immunohistochemical detection of infectious agents following prolonged formalin fixation. Therefore, the goal of this study was to evaluate the effects of prolonged formalin fixation on the immunohistochemical detection of 21 infectious agents. Tissue slices about 5 mm thick were fixed in 10% neutral-buffered formalin, processed, and paraffin embedded at day 1 or 2 and then at approximately weekly intervals. Three pathologists graded immunoreactivity according to a four-tier grading system: negative, weak, moderate, strong. Canine parvoviral immunoreactivity was markedly decreased following 2, 7, and 10 weeks of fixation in myocardium, small intestine, and spleen, respectively. Bovine respiratory syncytial virus immunoreactivity was markedly decreased following 7 weeks of fixation. Bartonella henselae had an abrupt loss of immunoreactivity following 9 weeks of fixation. Despite variation among time points, immunoreactivity remained moderate to strong throughout the study period for the other 18 antigens. These results suggest that prolonged formalin fixation of up to 7 weeks generally does not limit immunohistochemical detection of infectious agents. However, the effects of prolonged fixation depend on the targeted antigen and the selected antibody. The results of this study further validate the utility and reliability of immunohistochemistry in diagnostic pathology.