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Co-delivery of chemotherapeutic agents improve anti-tumor efficacy and reduce cancer resistance. Here, we report development of layer-by-layer assembled gold nanoparticles (LbL-AuNP) containing anti-STAT3 siRNA and imatinib mesylate (IM) to treat melanoma. The combination treatment with STAT3 siRNA and IM in B16F10 melanoma cells showed greater suppression of STAT3 protein, decreased cell viability and increased apoptotic events compared with LbL-AuNP containing either STAT3 siRNA or IM. In vivo efficacy studies in melanoma tumor bearing mice showed that non-invasive topical iontophoretic administration (0.5 mA/cm2) of LbL-AuNP was comparable with intratumoral administration. Co-delivery of STAT3 siRNA and IM using LbL-AuNP showed significant (p < 0.05) reduction in percentage tumor volume, tumor weight and suppressed STAT3 protein expression compared with either STAT3 siRNA or IM loaded LbL-AuNP. Taken together, LbL-AuNP can be developed as nanocarrier system for co-delivery of siRNA and small molecule drugs for topical iontophoretic delivery.