Nigrospora osmanthi strain Z1 is a potential bioherbicide for the biological control of water lettuce, one of the most common invasive weeds. Here, the first high-quality genome assembly of N. osmanthi was obtained by a combination of PacBio and Illumina sequencing technologies. Twelve contigs representing the nuclear genome and one mitochondrial DNA were generated, with a total length of 45.94 Mb, an N50 equal to 6.99 Mb, and a GC content of 57.91%; 9 of the 12 nuclear contigs represented chromosomes from telomere to telomere characterized by telomeric-like repeats. A total of 11,051 genes were predicted using a combination of gene prediction tools, including 336 noncoding RNAs, 1,298 genes encoding secretory proteins, 1,735 genes encoding carbohydrate-active enzymes, 1,955 fungal virulence factors, and 50 putative secondary metabolite gene clusters. This complete genome sequence can serve as a valuable resource for future comparative genomic studies of the fungus Nigrospora for effective application in the biocontrol of invasive species and the discovery of fungal secondary metabolites. [Figure: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.