For the first time, an ultralight chitosan-glutaraldehyde nanofiber sponge (chitosan NF sponge) was prepared. The present work describes its processing from pure electrospun chitosan nanofibers and its use for filtration applications. Chitosan/polyethylene oxide (PEO) nanofibers (NF) were electrospun from acetic acid into 309 ± 56 nm-thick nanofibers using high-throughput free-surface electrospinning. To yield chitosan NF sponges, PEO was extracted from the defect-free nanofiber mats. From these mats, nanofiber suspensions were prepared followed by casting and freeze-drying. Cross-linking of such obtained pristine chitosan NF sponges with glutaraldehyde improved water stability and resulted in chitosan NF sponges with a bulk density of 5.77 mg cm–3 and a porosity of 99.59%. The hierarchical pore architecture of the chitosan NF sponges was perfectly suited for particle adsorption as tested for poly(ethylene terephthalate)-microplastic (PET-MP) and Arizona test dust (ISO 12103-1) suspensions. Hydrostatic filtration with chitosan NF sponges reduced turbidity of particle suspensions by 99.46% nephelometric turbidity units (NTU) (PET-MP) and 99.49% (Arizona test dust). An oyster-inspired adsorption setup with 4000 actuated compression/relaxation cycles reduced the turbidity of PET-MP and Arizona test dust suspensions by 80.1 ± 1.5 and 91.9 ± 0.3% NTU, respectively. The preparation of biocompatible NF sponges from chitosan marine biomass has been demonstrated. These chitosan NF sponges can be used as efficient filters to tackle environmental challenges such as microplastics.