This study aimed to develop a Lactobacillus plantarum (L. plantarum)-loaded dual-layer wound dressing (DLD) with excellent wound recovery and mechanical properties. L. plantarum-loaded DLD was fabricated by covering the hydrogel (inner layer) with a hydrocolloid (external layer). The hydrocolloid was manufactured by the hot-melt method, consisting of liquid paraffin, polyisobutylene, styrene-isoprene-styrene, and sodium carboxymethylcellulose (12:20:25:43, w/w/w/w). In contrast, the hydrogel was fabricated by the freeze-and-thaw method to load heat-labile L. plantarum. Various non-ionic materials have been investigated to select appropriate hydrogel components. The hydrogel composed of L. plantarum stock solution, guar gum, and polyvinyl alcohol (10:2:10, w/w/w) was chosen for its excellent swelling capacity and mechanical properties. As a result, heat-labile L. plantarum was successfully loaded into the guar-gum-based DLD. Moreover, guar gum-based DLD containing L. plantarum exhibited significantly enhanced swelling capacity and elasticity compared to single hydrogel layer (swelling capacity: DLD, 920.7 ± 32.4 % vs. hydrogel, 282.2 ± 6.5 %; elastic modulus: DLD, 2.9 ± 0.3 × 10−3 N/mm2 vs. hydrogel, 4.2. ± 0.7 × 10−3 N/mm2). The wound recovery test using Pseudomonas aeruginosa-infected animal model and histological profiles confirmed guar gum-based DLD containing L. plantarum to elicit accelerated wound recovery with complete re-epithelialization compared to commercial product and non-treated (recovery rate at Day 3: DLD, 67.8 ± 6.2 % vs. commercial product, 30.4 ± 11.7 % vs. non-treated, 14.2 ± 7.5 %). Therefore, L. plantarum-loaded DLD is an effective system for wound treatment.