Nanoparticulate delivery systems have been noticed for chemotherapeutical delivery due to their ability in controlling the drug release and reducing the side effect. These systems could also be used to deliver two drugs or more simultaneously, inhibiting the development of resistant cancerous cells. Methotrexate (MTX), one of the most frequently used chemotherapeutic agent, and Curcumin (CUR), a natural chemopreventive compound, have shown promising results in treatment or controlling the progression of cancer. The aim of this study is to prepare and evaluate polymeric nanoparticles for co-delivery of MTX and CUR. The PLGA nanoparticles were prepared and characterized in respect of their particles size, morphology, drug encapsulation efficiencies, release patterns, cell cytotoxicity, and in vivo efficacy. Altering MTX and CUR amounts leads to particle size of 142.3 ± 4.07 nm with MTX encapsulation efficiency of 71.32 ± 7.8% and CUR encapsulation efficiency of 85.64 ± 6.3%. These particles showed significantly higher cytotoxicity in comparison with free MTX or CUR or even their solo-loaded formulations. The in vivo results showed the synergic effect of MTX and CUR co-delivery on inhibiting the progression of breast cancer. Considering the appropriate in vitro properties of acquired nanoparticles for controlled drug delivery and the satisfactory in vivo efficacy results, it seems that the prepared formulation is a promising candidate for further in vivo studies.