The resistance and impermeability of bacterial biofilms lead to incurable infections. Interference with bacterial respiration is the key to the eradication of bacterial biofilm, but breaking the deep-tissue biofilm barrier to disrupt bacterial respiration still lacks effective means. Here, we report a hydrogel microsphere that disrupts bacterial respiration, supports in situ production of carbon monoxide gas (CO) to enhance the oxygen-depleted environment of biofilms and disrupts the bacterial respiratory chain, eliminating the bacterial biofilm ecotone (BRDMs). Under the specific interaction of α-helical structure and bacterial biofilm, BRDMs rapidly anchored and accumulated on the surface of bacterial biofilm within 8 h. Meanwhile, 8.64 μM CO gas was released in situ under an oxidative stress environment to deeply penetrate the biofilm and continuously destroy bacterial terminal oxidase, block bacterial respiration and finally disintegrate the biofilm. In a model of osteomyelitis, BRDMs disrupt the ecotopic colonization of MRSA biofilms in deep tissues, reduce inflammation, restore internal environmental homeostasis and accelerate tissue regeneration. BRDMs could be designed to remove drug-resistant biofilms from a wide range of deep tissues. Display omitted •Interference with bacterial respiration is the key to the eradication of bacterial biofilm.•CO gas enhances the oxygen-depleted environment of biofilms and disrupts its respiratory to eliminate bacterial biofilm.•BRDMs disrupt the ecotopic colonization of biofilms in deep tissues of osteomyelitis and accelerate tissue regeneration.•BRDMs could be designed to remove drug-resistant biofilms from a wide range of deep tissues.