The aim of this study was to obtain a prolonged release of Vitamin A palmitate (RAP) and aciclovir from biodegradable microspheres for intraocular administration with an antiviral action and to be capable of preventing the inherent risks of intravitreal administration. The RAP effect on the microsphere characteristics was also studied. Poly(D,L-lactic-co-glycolic) acid microspheres were prepared by the solvent evaporation method. Different quantities of aciclovir (40-80 mg) and RAP (10-80 mg) were added to the internal phase of the emulsion. Microspheres were analysed by scanning electron microscopy, which revealed a spherical surface and a porous structure, and granulometric analysis that showed an adequate particle size for intraocular administration. The aciclovir loading efficiency increased when Vitamin A palmitate was added. Differential scanning calorimetry detected no differences in the polymer glass transition temperature and the aciclovir melting endotherm in all formulations. The release of aciclovir during the first days of the in vitro assay was improved with respect to microspheres without RAP. The microspheres showed a constant release of aciclovir and RAP for 49 days. Best results were obtained for microspheres prepared with 40 mg aciclovir, 80 mg RAP and 400mg polymer. A dose of 4.74 mg of microspheres would be therapeutic for the herpes simplex and Epstein-Barr viruses' treatment in an animal model and would reduce the intravitreal adverse effects. The injectability of a suspension of microspheres in isotonic saline solution resulted appropriate for its injection through a 27 G needle.