It is likely that phylogroup 2 lyssaviruses circulate within bat reservoirs. We adapted a pseudotype (pt) neutralisation assay (PNA) to a multiplex format enabling serosurveillance for Lagos bat virus (LBV), Mokola virus (MOKV) and West Caucasian bat virus (WCBV) in a potential reservoir, the African straw-coloured fruit bat, Eidolon helvum. Highly correlated titres were observed between single and multiplex PNAs using ptLBV and ptMOKV (r=0.97, p<0.0001), validating its use for bat serosurveillance. Of the bat serum samples screened 56% neutralised ptLBV, 27% ptMOKV and 1% ptWCBV. Mean VNAb titres were 1:266, 1:35 and 1:7 against ptLBV, ptMOKV and ptWCBV respectively. The high seroprevalence estimates suggest that the infection rate of LBV in E. helvum remains high enough to persist in this species. This supports the hypothesis that LBV is endemic in Ghanaian E. helvum and we speculate that LBV may have co-evolved with African megachiroptera.