•This is the first report of endophytic Biscogniauxia in Dendrobium species•Biscogniauxia dendrobii sp. nov. is introduced based on the large discrepancy on both sequences (5.2%) and size of conidia.•The ethyl acetate crude extract of the isolate B. petrensis showed moderate and strong cytotoxic activity against A549 and K562 using CCK8 assay (IC50 of it 13.5 and 3.5 ug/ml, respectively). Biscogniauxia belongs to the family Graphostromataceae, and species have been found mainly as pathogens in dicotyledonous plants worldwide. Two endophytic strains, Biscogniauxia dendrobii sp. nov. and B. petrensis were isolated from Dendrobium aphyllum and D. harveyanum sampled from southwestern China and northern Thailand respectively. The conidiophores and conidia were used for morphological examination. The maximum likelihood and Bayesian analysis with ITS, TUB2, RPB2 and LSU sequence data were combined for phylogenetic analyses and species identification. Biscogniauxia dendrobii sp. nov. is introduced based on the large DNA base pair differences (5.2%) and size of conidia. Another known species B. petrensis is also illustrated and described in this study to facilitate species identification on D. harveyanum. The ethyl acetate crude extracts of the isolate B. petrensis showed moderate and strong cytotoxic activity against A549 and K562 using the CCK8 assay (IC50 of it 13.5 and 3.5 ug/ml respectively). This is the first report of endophytic Biscogniauxia in Dendrobium species and of their cytotoxicity to A549 and K562, all of which enhances the current knowledge on the genus Biscogniauxia.