<p>Membrane separation provides an efficient alternative to alleviate water scarcity. However, it remains challenging to mitigate membrane fouling, especially biofouling, and surpass performance trade-off limitation. Here we report an antibiotic membrane with broad-spectrum antibacterial properties for highly permeable and selective water purification. Using the antibiotic kanamycin and trimesoyl chloride as monomers, a polyamide-polyester membrane was constructed through interfacial polymerization. This membrane exhibits competitive separation performance, with a high water permeance of 47.9 l m⁻² h⁻¹ bar⁻¹, solute rejection of 99.6% and solute–solute selectivity of ~10,000, outperforming most existing membranes. Moreover, this membrane can effectively inactivate Gram-negative/positive, single/multiple-resistant and disinfectant-resistant bacteria at high concentrations of 3 × 10⁷ colony-forming units per millilitre, showing mortality ratios of 93.6–99.9%. In addition, this membrane maintains long-term antibacterial durability during crossflow filtration for at least 170 h. These concepts and findings offer an alternative route to the design of high-performance and antifouling membranes for water treatment.</p>