Display omitted •A true-nanoscale PLA nanofiber with an average size of 37 ± 4 nm was fabricated.•The multi-scale structured nanofiber membrane was designed by one-step method, and the performance parameters were optimized.•The mask filter made of multi-scale structured nanofiber membrane showed high filtration efficiency and breathability.•The resultant mask filter has biodegradability and would be a great substitute for existing commercial masks in the future. The usage of single-use face masks (SFMs) has increased since the outbreak of the coronavirus pandemic. However, non-degradability and mismanagement of SFMs have raised serious environmental concerns. Moreover, both melt-blown and nanofiber-based mask filters inevitably suffer from poor filtration performance, like a continuous decrease in the removal efficiency for particulate matter (PM) and weak breathability. Herein, we report a new method to create biodegradable and reusable fibrous mask filters. The filter consists of a true nanoscale bio-based poly(lactic acid) (PLA) fiber (an average size of 37 ± 4 nm) that is fabricated via electrospinning of an extremely dilute solution. Furthermore, we designed a multiscale structure with integrated features, such as low basis weight (0.91 g m−2), small pore size (0.73 μm), and high porosity (91.72%), formed by electrospinning deposition of true nanoscale fibers on large pore of 3D scaffold nanofiber membranes. The resultant mask filter exhibited a high filtration efficiency (PM0.3–99.996%) and low pressure drop (104 Pa) superior to the commercial N95 filter. Importantly, this filter has a durable filtering efficiency for PM and natural biodegradability based on PLA. Therefore, this study offers an innovative strategy for the preparation of PLA nanofibers and provides a new design for high-performance nanofiber filters.