In this study, antimicrobial membranes based on biodegradable material poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-4HB)] and quaternary ammonium salts (QASs) by two methods have been performed. Three QASs with varied alkyl chain lengths have been synthesized successfully and characterized by H-1 nuclear magnetic resonance and Fourier transform infrared. The synthesized QASs were blended with P(3HB-4HB) and electrospun into composite fibrous membranes or casted into conventional membranes. Electrospun fibrous membranes with large surface areas are a superior type of antimicrobial biomaterials, and they exhibit preferable properties than solution casting membranes. Specifically, electrospun fibrous membranes are tougher and can inactivate both Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli O157:H7 in a contact time of 30min, whereas the solution casting membranes cannot. The length of alkyl chain in the quaternary ammonium groups on the modified P(3HB-4HB) membranes is able to influence the antimicrobial activity. This type of antimicrobial material may have potential applications in biomaterial field. Copyright (c) 2016 John Wiley & Sons, Ltd.