BACKGROUND: Bats and rodents have been identified as reservoirs for several highly pathogenic and zoonotic viruses including henipaviruses, a genus within the Paramyxoviridae family. A number of studies have revealed the circulation of henipaviruses at the wildlife-human-livestock interface in Cameroon. In this study, we describe the molecular analysis as well as the development and evaluation of a Bead-based Multiplex Binding Assay (BMBA) using an in-house Indirect Enzyme Linked Immunosorbent Assay (ELISA) to confirm the detection of henipavirus infection in wildlife species. RESULTS: A total of 600 fruit bats and 600 rodents were sampled between March 2018 and June 2020. Samples were analyzed using a semi-nested RT-PCR assay followed by sequencing of the PCR fragments. Transudates (754) were screened for the presence of henipavirus-specific antibodies in a BMBA and confirmed by ELISA using Hendra virus (HeV), Nipah virus (NiV) and Ghana virus (GhV) glycoproteins expressed in Leishmania tarentolae, and commercially available HeV G and NiV G glycoproteins. Henipavirus-specific antibodies were detected in 19/531 (3.6%) bat transudates screened by BMBA and confirmed by ELISA. Seroprevalence rates in the Centre and North Regions were 12/291 (4.1%) and 7/240 (2.9%) respectively. All rodents and shrews were serologically negative. Henipavirus RNA sequences were not detected in any of the samples screened in this work. CONCLUSION: This study provides further data supporting the circulation of Henipaviruses in fruit bats (Eidolon helvum) which are roosting and reproducing in proximity to human and livestock populations in the Centre and North Regions of Cameroon. This also establishes the first detection of Henipavirus specific antibodies in Eidolon helvum populations in the North Region of Cameroon.