Background The utilization of zein and gum arabic has grown in an attempt to formulate wall materials based on protein–polysaccharide complexes. This mixture provides a versatile shelter for hydrophilic (guava leaf extract, GLE) or lipophilic (fish oil, FO) bioactive compounds from unwanted environmental factors, and it can be used as an edible film-forming polymer. This study was undertaken to characterize FO-containing edible films that were double-layered with a film containing GLE. Methods Modified zein and gum arabic solutions (MG complex) were mixed at a ratio of 1:1.5 (v/v), adjusted to pH 5, added with glycerol (20% of the complex) and FO (5% of the complex), and finally adjusted to pH 5. This was prepared as the bottom/lower layer. The upper/active layer was prepared by mixing MG complex, glycerol, and GLE (1, 3, and 5% w/v of the complex). Physical, mechanical, microstructural, thermal, microbiological, and oxidative measurements were also performed. Results The total phenolic and flavonoid contents in GLE were 15.81 mg GAE/g extract and 6.99 mg QE/g extract, respectively. The IC50 of the DPPH radical scavenging activity of GLE was 26.86 ppm with antibacterial activity against Bacillus subtilis and Escherichia coli of 9.83 and 12.55 mm. The total plate counts of films double-layered with a film containing GLE were retained below 3 log CFU/g during 28-day storage. The peroxide values of these films were dimmed for no more than 9.08 meq/kg sample on day 28 of storage. Thickness (872.00-971.67 μm), water vapor transmission rate (12.99-17.04 g/m2/day), tensile strength (1.56-2.02 kPa), elongation at break (61.53-75.41%), glass transition (52.74-57.50°C), melting peak (131.59-142.35°C), inhibition against B. subtilis (33.67-40.58 mm), and inhibition against E. coli (2.05-9.04 mm) were obtained by double-layer films. Conclusions GLE can be successfully incorporated into the active layer of a double-layer film to improve its characteristics while significantly slowing down the microbial contamination and oxidation rate.