The fungal genus Nigrospora is known to be a plant pathogen, endophyte, and saprobe, and it is usually isolated from various substrates like soil and air. During the surveys of soil fungi in Hebei Province of China, two isolates of Nigrospora were obtained. A multi-locus phylogeny of combined loci of the 5.8S nuclear ribosomal gene with the two flanking transcribed spacers (ITS), part of the translation elongation factor 1-alpha (tef1), and the beta-tubulin (tub2) loci, in conjunction with morphological characters were used to identify the newly collected isolates. Nigrospora humicola sp. Nov. is described and proposed herein, which differs from its phylogenetically close species N. chinensis and N. globosa by the sequences of ITS, tef1, and tub2.