Safe and efficient antibacterial materials are urgently needed to combat drug-resistant bacteria and biofilm-associated infections. The rational design of nanoparticles for flexible elimination of biofilms remains challenging. Herein, we propose the fabrication of Janus-structured nanoparticles targeting extracellular polymeric substance to achieve dispersion or near-infrared (NIR) light-activated photothermal elimination of drug-resistant biofilms, respectively. Asymmetrical Janus-structured dextran-bismuth selenide (Dex-BSe) nanoparticles are fabricated to exploit synergistic effects of both components. Interestingly, Janus Dex-BSe nanoparticles realize enhanced dispersal of biofilms over time. Alternatively, taking advantage of the preferential accumulation of nanoparticles at infection sites, the self-propelled active motion induced by the unique Janus structure enhances photothermal killing effect. The flexible application of Janus Dex-BSe nanoparticles for biofilm removal or NIR-triggered eradication in vivo is demonstrated by Staphylococcus aureus-infected mouse excisional wound model and abscess model, respectively. The developed Janus nanoplatform holds great promise for the efficient elimination of drug-resistant biofilms in diverse antibacterial scenarios.