Otitis Media (OM) is the most common reason for U.S. children to get prescribed antibiotics. Current oral multidose antibiotics have the potential for causing a range of side effects and development of antimicrobial resistance (AMR) due to systemic antibiotic exposure. Developing innovative antimicrobials with single-dose delivery and sustained efficacy is an effective strategy to circumvent these negative impacts of oral antibiotics. To achieve this goal, we synthesized vanadium pentoxide nanowires (V2O5) which could turn metabolic products from pathogens into a potentantimicrobial, hypohalous acid. This system was applied to the OM treatment in a chinchilla model. Single-dose V2O5 formulation was given through chinchilla’s bullae and could completely eradicate OM from Streptococcus pneumoniae in the middle ear in 7 days. In addition, no tissue toxicity was observed at the given dose of V2O5 formulation. Nevertheless, due to the micrometer length of the V2O5 nanowires, the formulation had to be delivered via intratympanic or intrabullae injections. To further achieve non-invasive transtympanic delivery, we developed sub-10-nm silver nanoparticles, stabilized with polyvinylpyrrolidone (AgNPs-PVP), with the potential of penetrating intact tympanic membranes. AgNPs-PVP showed high antimicrobial efficacy against the two main OM pathogens, namely non-typeable Haemophilus influenzae and Streptococcus pneumoniae. To ensure sustained presence of AgNPs-PVP in the middle ear, a formulation based on poloxamer 407 (P407) was designed such that an AgNPs-containing solution can be administered through the tympanic membrane, which then gels quickly into a firm hydrogel, delivering prolonged antimicrobial effect. Future research will focus on surface functionalization of the AgNPs with chemical permeation enhancer to enable trans tympanic delivery of nanoparticles.