Objective To evaluate the usefulness of nested-polymerase chain reaction (nested-PCR), indirect fluorescent antibody assay (IFA), immunosuppressive provocation tests, and histopathological diagnosis assays for detecting Clostridum Piliforme (C. Piliforme) infections in laboratory rats. Methods In this study, the nested-PCR primers, which we synthesized to amplify C. Piliforme 16S ribosomal DNA (rDNA) sequences as an identification system for this organism, were performed by using C. Piliforme and 15 non-C. Piliforme strains. Specimens taken from 20 immunosuppressive Wistar rats and 5 non-immunosuppressive SD rats and submitted to detect C. Piliforme by nested-PCR. Results The C. Piliforme reference strain revealed positive result indicated by a 196 bp specific fragment on gel, while 15 non-C. Piliforme standard strains gave negative results demonstrating that the inner primers should be specific for C. Piliforme tested. The nested-PCR would be able to detect as little as 10 pg C. Piliforme-DNA. Tissues samples taken from rats and submitted to detect C. Piliforme by nested-PCR were found negative results, and the results of nested-PCR and consistent with those of conventional bacteria identification tests and histopathological diagnosis assays. 25 serum samples taken from rats were submitted to detect C. Piliforme antibodies by IFA were found heterologous cross-reactive antigens such as some strains of Clostridia and Bacillus. Conclusion IFA is best used for colony surveillance, and follow-up testing may include immunosuppressive provaction tests. Confirmation of C. Piliforme infections is best performed by PCR, bacteria identification tests, and histopathological diagnosis assays. In our study, the results of nested-PCR are consistent with those of bacteria identification tests and histopathological diagnosis assays. The data have also demonstrated that the nested-PCR established here is a quick, reliable, sensitive and specific method for detecting C. Piliforme infections in laboratory rats.