As problems of antibiotic resistance increase, a continuing need for effective bioactive wound dressings is anticipated for the treatment of infected chronic wounds. Naturally derived antibacterial agents, such as Manuka honey, consist of a mixture of compounds, more than one of which can influence antimicrobial potency. The non-peroxide bacteriostatic properties of Manuka honey have been previously linked to the presence of methylglyoxal. The incorporation of methylglyoxal as a functional antibacterial additive during fibre production was explored as a potential route for manufacturing wound dressing components. Synthetic methylglyoxal and poly(vinyl alcohol) were fabricated into webs of sub-micron fibres by means of electrostatic spinning of an aqueous spinning solution. Composite fabrics were also produced by direct deposition of the poly(vinyl alcohol)–methylglyoxal fibres onto a preformed spunbonded nonwoven substrate. Attenuated total reflectance fourier transform infrared and proton nuclear magnetic resonance spectroscopies confirmed the presence of methylglyoxal within the resulting fibre structure. The antibacterial activity of the fibres was studied using strains of Staphylococcus aureus and Escherichia coli. Strong antibacterial activity, as well as diffusion of methylglyoxal from the fibres was observed at a concentration of 1.55 mg/cm2.