Viral-bacterial coinfection is a major and life-threatening issue, and it ranks among the primary causes of mortality. Current monotherapies that only target either bacteria or viruses, are deemed insufficient for treating coinfection and may be associated with undesirable consequences. Therefore, the development of novel treatment approaches is necessary, preferably with dual effects against both viruses and bacteria. This study sought to assess the effectiveness of PLNC8 αβ, in specifically targeting both KUNV and S. aureus infection in an in vitro coinfection model using human keratinocyte cells. The LDH cytotoxicity test was performed to evaluate the peptide’s effectiveness in reducing cytotoxicity that is individually caused by S. aureus, KUNV, as well as their coinfection. Additionally, ELISA was utilized to quantify the levels of inflammatory cytokines, namely CXCL8, and IL-6. The results demonstrated that both forms of the PLNC8 αβ effectively decreased the infection-induced cellular cytotoxicity. The D- PLNC8 αβ exhibited superior efficacy compared to the L- PLNC8 αβ, since the latter was more susceptible to enzymatic degradation, resulting in the loss of its functionality. Furthermore, both forms of PLNC8 αβ effectively modulated the levels of inflammatory cytokine and restored cellular viability. In addition, the peptide substantially reduced the number of bacterial colonies in both S. aureus infection and coinfection. Based on these findings D- PLNC8 αβ possesses a dual antimicrobial action and could be further characterized and validated as a promising therapeutic agent against viral-bacterial coinfection.