In this study, the electrospinning technique was used to co-encapsulate Quercetin (Qu) and Lactiplantibacillus plantarum 1–24-LJ in PVA-based nanofibers, and the effect of bioactive films on fish preservation was evaluated at the first time. The findings indicated that both Lpb. plantarum 1–24-LJ and Qu were successfully in the fibers, and co-loaded fibers considerably outperformed single-loaded fiber in terms of bacterial survival and antioxidant activity. Following fish preservation using the loaded fibers, significant reductions were observed in TVB-N, TBARS, and microbial complexity compared to the control group. Additionally, the co-loaded fibers more effectively reduced the counts of H2S-producing bacteria and Pseudomonas. In the future, fibers with both active substances and LAB hold promise as a novel approach for fish preservation.