Biodegradable polymers such as poly lactide-co-glycolides (PLGA) have been considered for the preparation of nanoparticles (NPs). In this study, rifampicin (RIF)-loaded PLGA NPs were fabricated by an emulsification/solvent diffusion method. The effect of several variables on the NPs' characteristics were evaluated, including the amount of RIF, amount of the poly vinyl alcohol as surfactant, and internal-phase volume and composition. The RIF encapsulation efficacy and the particle size distribution were optimized by varying these parameters. NPs were spherical with a relatively monodispersed size distribution. The effect of nanoencapsulation of RIF on the antibacterial activity of RIF against gram-positive and gram-negative bacteria was evaluated. It was shown that RIF NPs could considerably improve the RIF antibacterial efficacy.