Biofilms are responsible for persistent or recurring microbial infections. Polymicrobial biofilms are prevalent in environmental and medical niches. Dual-species biofilms formed by Gram-negative uropathogenicEscherichia coli(UPEC) and Gram-positiveStaphylococcus aureusare commonly found in urinary tract infection sites. Metal oxide nanoparticles (NPs) are widely studied for their antimicrobial and antibiofilm properties. We hypothesized that antimony-doped tin (IV) oxide (ATO) NPs, which contain a combination of antimony (Sb) and tin (Sn) oxides, are good antimicrobial candidates due to their large surface area. Thus, we investigated the antibiofilm and antivirulence properties of ATO NPs against single- and dual-species biofilms formed by UPEC andS. aureus. ATO NPs at 1 mg/mL significantly inhibited biofilm formation by UPEC,S. aureus, and dual-species biofilms and reduced their main virulence attributes, such as the cell surface hydrophobicity of UPEC and hemolysis ofS. aureusand dual-species biofilms. Gene expression studies showed ATO NPs downregulated thehlagene inS. aureus, which is essential for hemolysin production and biofilm formation. Furthermore, toxicity assays with seed germination andCaenorhabditis elegansmodels confirmed the non-toxic nature of ATO NPs. These results suggest that ATO nanoparticles and their composites could be used to control persistent UPEC andS. aureusinfections.