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Abstract Electrospun fish gelatin (FGel) nanofibers (NF) mimic the natural bodies extracellular matrix's (ECM) structure and are an attractive material for many biomedical applications. However, FGel poor mechanical properties and rapid dissolution in an aqueous media paired with usually low productivity of the typical electrospinning process necessitate further effort in overcoming these issues. In this study, alternating field electrospinning (AFES) fabricates cold water fish skin gelatin nanofibrous materials (FGel NFM) with up to 10 wt.% Dextran (DEX) or acetyl glucosamine (AGA) from pure aqueous solutions at process productivity of 7.92–8.90 g∙h⁻¹. Thermal crosslinking of as‐spun materials resulted in FGel‐based NFM with 125–325 nm fiber diameters. DEX (MW500k and MW75k) and AGA additives cause different effects on FGel fiber diameters, structure, tensile and degradation behavior, and in vitro performance. All tested materials reveal favorable, but not the same, cellular response through the formation of a confluent layer on the NFM surface regardless of the fibers’ composition despite the significant difference in FGel NFM structure and properties. Results show that AFES and thermal crosslinking of FGel‐based NFM can lead to a sustainable “green” fabrication technology of mono‐ and polysaccharide modified FGel‐based NFM scaffolds with the parameters attuned to targeted biomedical applications.