Conversion of bio-based industrial residues into high value-added products such as natural nanofibers is advantageous from an environmental and economic perspective, promoting resource efficiency along with the utilization of renewable materials. However, in order to employ the benefits of the raw material; its eco-efficient production should further be developed. Within this context, eco-design optimization through life cycle assessment (LCA) combined with life cycle costing (LCC) were applied to target eco-efficient production of natural nanofibers from carrot residue, along with quality assessment. The initial production steps included pretreatment combined mechanical nanofibrillation via ultrafine grinding, where the largest contributors to the environmental impact were identified as chemicals and energy. These were targeted by omitting the alkali pretreatment step and instead applying direct bleaching prior to nanofibrillation. After eco-design optimization, the yield increased while the energy, chemical, and water use significantly decreased. Therefore, a reduced environmental impact of more than 75% each for carbon footprint, freshwater ecotoxicity, and human toxicity was shown, along with a cost reduction of more than 50%. The use of carrot residue displayed an efficient conversion into natural nanofibers that was further promoted with the use of eco-design, yet with sustained functionality and nanoscaled dimensions, thus promoting resource-efficiency and natural nanofiber implementation in a wide range of promising bio-based applications.