The otoprotective effects of OTO-104 were investigated both prior to and following acute acoustic trauma. Guinea pigs received a single intratympanic injection of OTO-104 and were assessed in a model of acute acoustic trauma. Doses of at least 2.0% OTO-104 offered significant protection against hearing loss induced by noise exposure when administered 1 day prior to trauma and up to 3 days thereafter. Otoprotection remained effective even with higher degrees of trauma. In contrast, the administration of a dexamethasone sodium phosphate solution did not protect against noise-induced hearing loss. Activation of the classical nuclear glucocorticoid and mineralocorticoid receptor pathways was required for otoprotection by OTO-104. The sustained exposure properties of OTO-104 were also superior to a steroid solution.