Oxabornyl polyenes represent a unique group of polyketides characterized by a central polyene core flanked by a conserved oxabornyl moiety and a structurally diverse oxygen heterocyclic ring. They are widely distributed in fungi and possess a variety of biological activities. Due to the significant spatial separation between the two stereogenic ring systems, it is difficult to establish their overall relative configurations. Here, we isolated three oxabornyl polyenes, prugosenes A1–A3 (1–3), from Talaromyces sp. JNU18266-01. Although these compounds were first reported from Penicillium rugulosum, their overall relative and absolute configurations remained unassigned. By employing ozonolysis in combination with ECD calculations, we were able to establish their absolute configurations, and additionally obtained seven new chemical derivatives (4–10). Notably, through NMR data analysis and quantum chemical calculations, we achieved the structural revision of prugosene A2. Furthermore, prugosenes A1–A3 exhibited potent antiviral activity against the respiratory syncytial virus, with compound 1 displaying an IC50 value of 6.3 μM. Our study thus provides a valuable reference for absolute configuration assignment of oxabornyl polyene compounds.