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We report a facile and environment-friendly finishing method for cotton fabrics using zwitterionic sulfopropylbetaine(ISB) and quaternary ammonium salt (IQAS) with reactive isocyanate group (NCO) as antimicrobial finishing agents via dipping–padding–drying process. The results showed that ISB and IQAS can be covalently bound onto the cotton fabric surface, imparting exellent antifouling activity and bactericidal activity, respectively. The dually finished cotton fabrics exhibited excellent bactericidal and antifouling performance without significantly affect their water vapor permeability and air permeability. All the finished cotton fabrics exhibited improved breaking strength, tearing strength, bursting strength and hydrophilicity compared to raw cotton fabrics. Atomic force microscope research results indicated that the adhesion force between bacterial and finished cotton fabric surfaces increased with the QAS content, and they can be tailored by changing the ISB/IQAS ratio. Most importantly, non-leaching antimicrobial and antifouling cotton fabrics did not induce skin stimulation and toxic effects to animals because ISB and IQAS were chemically bound to cotton fabrics, ensuring that our finishing method have great potential for industrial and healthcare applications, including but not limited to fabrics, surgical equipment water purification systems, packaging and public health settings.