BackgroundMarine deep subsurface sediments were once thought to be devoid of eukaryotic life, but advances in molecular technology have unlocked the presence and activity of well-known closely related terrestrial and marine fungi. Commonly detected fungi in deep marine sediment environments includesPenicillium,Aspergillus,Cladosporium,Fusarium, andSchizophyllum, which could have important implications in carbon and nitrogen cycling in this isolated environment. In order to determine the diversity and unknown metabolic capabilities of fungi in deep-sea sediments, their genomes need to be fully analyzed. In this study, twoPenicilliumspecies were isolated from South Pacific Gyre sediment enrichments during Integrated Ocean Drilling Program Expedition 329. The inner gyre has very limited productivity, organic carbon, and nutrients.ResultsHere, we present high-quality genomes of two proposed novelPenicilliumspecies using Illumina HiSeq and PacBio sequencing technologies. Single-copy homologues within the genomes were compared to other closely related genomes using OrthoMCL and maximum-likelihood estimation, which showed that these genomes were novel species within the genusPenicillium. We propose to name isolate SPG-F1 asPenicillium pacificasedimentisp. nov. and SPG-F15 asPenicillium pacificagyrussp. nov. The resulting genome sizes were 32.6 Mbp and 36.4 Mbp, respectively, and both genomes were greater than 98% complete as determined by the presence of complete single-copy orthologs. The transposable elements for each genome were 4.87% forP.pacificasedimentiand 10.68% forP.pacificagyrus. A total of 12,271 genes were predicted in theP.pacificasedimentigenome and 12,568 genes inP.pacificagyrus. Both isolates contained genes known to be involved in the degradation of recalcitrant carbon, amino acids, and lignin-derived carbon.ConclusionsOur results provide the first constructed genomes of novelPenicilliumisolates from deep marine sediments, which will be useful for future studies of marine subsurface fungal diversity and function. Furthermore, these genomes shed light on the potential impact fungi in marine sediments and the subseafloor could have on global carbon and nitrogen biogeochemical cycles and how they may be persisting in the most energy-limited sedimentary biosphere.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12864-023-09320-6.