Purpose: This study aims to prepare a novel, natural nanoparticle (NP) as a drug carrier, which also has inherent therapeutic effects. Methods: Pistacia khinjuk gum NPs were prepared and Response surface methodology (RSM) was used for statistical analysis of data and optimizing the size of NPs. Results: NPs were in the range of 75.85–241.3 nm. The optimization study was carried out, and an optimized size (70.86nm) was obtained using DMSO as a solvent. The volume of the organic phase was 111.25µl, and the concentration of gum was 1% w/v. The cell viability assay was performed on the pure gum and NPs toward β-TC3, MCF7, and HT29 cell lines. It was observed that NPs have higher cytotoxic activity in comparison with pure gum, and that the IC50value was achieved at 1% of NPs in β-TC3 cells. The obtained NPs demonstrated antibacterial activity against two bacterial strains (Pseudomonas aeruginosa and Staphylococcus aureus). Conclusion: Altogether, according to the obtained results, these NPs with inherent cytotoxicity and antibacterial activity are an attractive carrier for drug delivery.