During an ongoing research survey of saprobic fungi in Thailand, four coelomycetous strains were isolated from decaying leaves in Chiang Mai and Phitsanulok Provinces. Morphological characteristics demonstrated that these taxa are typical of Cytospora in forming multi-loculate, entostromatic conidiomata, branched or unbranched conidiophores, with enteroblastic, phialidic conidiogenous cells and hyaline, allantoid, aseptate conidia. Multiloci phylogeny of ITS, LSU, ACT, RPB2, TEF1-α and TUB2 confirmed these taxa are distinct new species in Cytospora in Cytosporaceae (Diaporthales, Sordariomycetes), viz., Cytospora chiangmaiensis, C. phitsanulokensis and C. shoreae. Cytospora chiangmaiensis has a close phylogenetic relationship with C. shoreae, while C. phitsanulokensis is sister to C. acaciae. These three novel species were also preliminary screened for their antagonistic activity against five plant pathogenic fungi: Colletotrichum fructicola, Co. siamense, Co. artocarpicola, Co. viniferum and Fusarium sambucinum. Cytospora shoreae and C. phitsanulokensis showed >60% inhibition against Co. viniferum and F. sambucinum, while C. chiangmaiensis had moderate inhibition activity against all pathogens.