Acute respiratory tract infections (ARTI) are a leading cause of morbidity and mortality in infants worldwide. Considering the emergence of antimicrobial resistance as a global threat, there is increasing interest in immunomodulatory strategies to prevent respiratory infections. Since ARTIs are caused by several pathogens, immunomodulatory strategies aiming to engage innate responses represent a promising strategy to prevent ARTIs. Here, innate-stimulating nanoparticles (NPs) synthesized from combinations of polyanhydride copolymers and pattern recognition receptor (PRR) agonists were developed to increase disease resistance by activating innate mechanisms at the mucosal level. In vitro analysis on human and bovine respiratory epithelial cells showed that innate-sensor agonist-loaded NPs triggered transcription of inflammatory, antiviral, and antimicrobial mediators. Moreover, pre-treatment with NPs reduced human and bovine orthopneumovirus (RSV) infectious titers in vitro. Intranasal administration of PRR-containing polyanhydride NPs to mice led to transient production of cytokines and chemokines in lungs, suggesting immune activation. The immunogenicity and antiviral properties of NPs were dependent on both polyanhydride copolymer chemistry and the innate agonist encapsulated within the NPs. Prophylactic administration of NPs containing either TLR2/1, TLR4, or TLR2/7 agonists resulted in reduced RSV morbidity and viral lung loads. Selected NPs also showed protective effects when administered 14 days before infection. These results indicate that NPs efficiently prime human and bovine respiratory tract epithelial cells and trigger antiviral defenses in vitro and reduce RSV disease in mice. STATEMENT OF SIGNIFICANCE: Our research focuses on the use of polyanhydride nanoparticles (NPs) encapsulating innate sensor agonists to activate epithelial and airway cells. This innovative approach leverages the unique properties of nanotechnology to harness the innate immune system's potential, providing broad resistance against multiple pathogens. We designed a panel of PRR agonist-loaded polyanhydride NPs with varying chemistries and investigated their effectiveness as innate immunomodulators in the respiratory tract. We demonstrate that NPs activate protective innate immune responses in airway epithelial cells and reduce RSV infectious titers in vitro. NP-treated mice showed protection against RSV-induced morbidity and had reduced viral loads. These findings highlight the potential of polyanhydride NPs as a versatile platform for prophylactic intervention against respiratory viruses in both humans and livestock.