Manuka honey (MH) is used as an antibacterial agent in bioactive wound dressings via direct impregnation onto a suitable substrate. MH provides unique antibacterial activity when compared with conventional honeys, owing partly to one of its constituents, methylglyoxal (MGO). Aiming to investigate an antibiotic-free antimicrobial strategy, we studied the antibacterial activity of both MH and MGO (at equivalent MGO concentrations) when applied as a physical coating to a nonwoven fabric wound dressing. When physically coated on to a cellulosic hydroentangled nonwoven fabric, it was found that concentrations of 0.0054 mg cm−2 of MGO in the form of MH and MGO were sufficient to achieve a 100 colony forming unit % bacteria reduction against gram-positive Staphylococcus aureus and gram-negative Klebsiella pneumoniae, based on BS EN ISO 20743:2007. A 3- to 20-fold increase in MGO concentration (0.0170–0.1 mg cm−2) was required to facilitate a good antibacterial effect (based on BS EN ISO 20645:2004) in terms of zone of inhibition and lack of growth under the sample. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) was also assessed for MGO in liquid form against three prevalent wound and healthcare-associated pathogens, i.e., Staphylococcus aureus, gram-negative Pseudomonas aeruginosa and gram-positive Enterococcus faecalis. Other than the case of MGO-containing fabrics, solutions with much higher MGO concentrations (128 mg L−1–1024 mg L−1) were required to provide either a bacteriostatic or bactericidal effect. The results presented in this study therefore demonstrate the relevance of an MGO-based coating as an environmentally friendly strategy for the design of functional dressings with antibiotic-free antimicrobial chemistries.