The development of advanced facemasks stands out as a paramount priority in enhancing healthcare preparedness. In this work, different polypropylene non-woven fabrics (NWF) were characterised regarding their structural, physicochemical and comfort-related properties. The selected NWF for the intermediate layer was functionalised with zinc oxide nanoparticles (ZnO NPs) 0.3 and 1.2wt% using three different methods: electrospinning, dip-pad-dry and exhaustion. After the confirmation of ZnO NP content and distribution within the textile fibres by morphological and chemical analysis, the samples were evaluated regarding their antimicrobial properties. The functionalised fabrics obtained via dip-pad-dry unveiled the most promising data, with 0.017 ± 0.013wt% ZnO NPs being mostly located at the fibre’s surface and capable of total eradication of Staphylococcus aureus and Escherichia coli colonies within the tested 24 h (ISO 22196 standard), as well as significantly contributing (**** p < 0.0001) to the growth inhibition of the bacteriophage MS2, a surrogate of the SARS-CoV-2 virus (ISO 18184 standard). A three-layered structure was assembled and thermoformed to obtain facemasks combining the previously chosen NWF, and its resulting antimicrobial capacity, filtration efficiency and breathability (NP EN ISO 149) were assessed. The developed three-layered and multiscaled fibrous structures with antimicrobial capacities hold immense potential as active individual protection facemasks.