Chronic wound healing is associated with prolonged elevated inflammation and high levels of oxidative stress leading to cell death. The majority of wounds are colonized with antibiotic-resistant bacterial biofilms such as Pseudomonas aeruginosa and Staphylococcus aureus. An ideal wound treatment should include agents with antioxidant, anti-inflammatory, and antibiofilm behavior. Therefore, in this study, a combination of curcumin nanoparticle (Cur-NP) and silver nanoparticle (AgNP) (Cur-NP/AgNP) loaded PVA hydrogel was used to inhibit the bacterial attachment and subsequent biofilm formation of P. aeruginosa and S. aureus. Cur was known for its antioxidant and anti-inflammatory effect while being non-toxic to cells. Meanwhile, AgNP demonstrated superior anti-bacterial and antibiofilm activities against both P. aeruginosa and S. aureus. Cur-NP/AgNP loaded PVA hydrogels completely inhibited the bacterial attachment and biofilm formation, possibly due to synergistic effect of Cur-NPs and AgNPs in killing the bacterial cells. It should be highlighted that no surviving bacterial cells were noted for Cur-NP/AgNP loaded hydrogels. On the other hand, AgNPs or Cur-NPs alone loaded hydrogels were unable to achieve complete inhibition of biofilm formation, even though significant reduction in the biofilm mass was noted compared with control samples. Cur-NP and AgNP exerted oxidative-stress induced cell death in HaCaT cells via mitochondrial dysfunction, mitochondrial membrane potential (MMP) reduction, adenosine triphosphate inhibition, and increased cytochrome C release. The toxicity of formulation followed the decreasing trend: Cur-NP/AgNP <
AgNPs alone <
Cur-NPs alone. Taken together, the combination of Cur-NP/AgNP completely inhibited bacterial biofilm formation through bactericidal effect on the planktonic cells while exerted the least toxic effects towards skin cells.