Species within Tetraplosphaeriaceae have been frequently documented in recent years with the extensive investigations of microfungi along a latitudinal gradient from north to south in the Asian/Australian region. Both bamboo substrates and freshwater habitats serve as extensive reservoirs, hosting a rich diversity of fungi that exhibit broad geographical distributions. The most common fungi in these two environments are generally distributed in distinct families. However, our statistics have revealed an intriguingly distinct preference of Tetraplosphaeriaceae species for inhabiting both bamboo substrates and freshwater habitats. The genera (100%) and (100%) exhibit a strong preference, followed by (71%) and (67%). Our taxonomic and phylogenetic study of microfungi in southern China have identified four additional novel species, viz., sp. nov., sp. nov., sp. nov., and sp. nov. from bamboo substrates and freshwater habitats. In addition, has previously been documented from freshwater habitats in Thailand; however, we have once again isolated this species from decaying bamboo substrates in Guangdong, China. The new findings substantiate our hypothesis that the preference of Tetraplosphaeriaceae species for colonizing bamboo substrates and freshwater habitats will be more evident through more extensive investigations conducted in such environments.