We report the controlled release of the antibiotic tetracycline (Tet) from triple-layered (3L) electrospun matrices consisting of zein or a zein/PCL blend, where the drug was loaded into the central layer with the two outer layers acting as diffusion barriers. These fibrous matrices successfully encapsulated Tet and efficiently inhibited the growth of a clinical isolate, the methicillin-resistant Staphylococcus aureus strain MRSA252, as demonstrated in a modified Kirby–Bauer disc assay over 5 days. Whilst untreated zein fibres are unstable in an aqueous environment, rapidly shrinking due to plasticisation and film formation, blending zein with PCL stabilised the electrospun matrices and prevented them from shrinking. These 3L formulations display sustained antibiotic release and provide a proof of concept for zein-based polymeric matrices as wound dressings to treat or prevent bacterial infection. This is the first demonstration of the controlled release of a clinically used antibiotic from electrospun zein-based matrices.