The demand for ever-lighter structures raises the interest in bonding as a joining method, especially for materials that are difficult to join with traditional welding and bolting techniques. Structural adhesives, however, are susceptible to defects, but can be toughened in several ways: by changing their chemical composition or by adding fillers, even of nanometric size. Nanomaterials have a high surface area and limited structural defects, which can enhance the mechanical properties of adhesives depending on their nature, quantity, size, and interfacial adhesion. This work analyzes the Mode I fracture toughness of joints bonded with METLBOND® 1515-4M epoxy film and XantuLayr electrospun XD 10 polyamide nanofibers. Two joint configurations were studied, which differed according to the position of the nanomat within the adhesive layer: one had the nanofibers at the substrate/adhesive interfaces, and the other had the nanofibers in the center of the adhesive layer. Double cantilever beam joints were manufactured to evaluate the Mode I fracture toughness of the bonding with and without nano-reinforcement. The nanofibers applied at the substrate/adhesive interface improved the Mode-I fracture toughness by 32%, reaching the value of 0.55 N/mm. SEM images confirm the positive contribution of the nanofibers, which appear stretched and pulled out from the matrix. No fracture toughness variation was detected in the joints with the nanofibers placed in the middle of the adhesive layer.