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• The double-layer furcellaran/gelatin hydrolysate and Ala-Tyr dipeptide film were developed. • The addition of peptide improved antioxidant and properties of the film. • The properties of films were tested on fish carcasses during frozen storage. • The application of films on the fish did not significantly inhibit oxidation rate. This is the first time that active double-layered furcellaran/gelatin hydrolysate films containing Ala-Tyr peptide were developed and characterised for their properties. Afterwards, films were used on Atlantic mackerel stored at −18 °C for 4 months and samples were analysed for changes in their microbiological quality, TVB-N, biogenic amine content, fatty acid composition and TBARS. Active films had higher TS (13.4 MPa) and lower WS (62.8%). The films showed no DPPH radical scavenging properties but high FRAP (6.6 mMol Trolox/mg). No significant effects on the oxidation of fish samples were observed with TBARS increasing from 12.04 to 22.50 mg/kg. Freezing successfully inhibited the growth of microorganisms and no differences in microbiological growth or biogenic amine formation were observed. However, the application of films inhibited the formation of TVB-N. Antimicrobiological properties of the film should be further investigated during storage of perishable food products at temperatures above 0 °C. [ABSTRACT FROM AUTHOR]