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In the last decade, the application of non-<i>Saccharomyces</i> yeasts in oenology as a natural tool to obtain wine diversification and higher quality has aroused great interest. In this work, three <i>Metschnikowia pulcherrima</i> strains, isolated from a vineyard, were characterised through the evaluation of their main oenological properties, antimicrobial activity, and specific enzymatic activities (β-glucosidase, β-lyase, polygalacturonase, and protease). The <i>M. pulcherrima</i> strains did not produce any inhibition against <i>Saccharomyces cerevisiae</i>, while they were able to exert an antimicrobial action against some unwanted bacteria and yeasts frequently present in grape must and potentially causing the alteration of wines. After this preliminary screening, <i>M. pulcherrima</i> AS3C1 has been selected to be used in the winemaking of red grape <i>Vitis vinifera</i> cv. Aglianico on a pilot scale. The effect of the sequential inoculation of <i>M. pulcherrima</i> AS3C1 with a commercial strain of <i>S. cerevisiae</i> was verified using for comparison a single inoculum with <i>S. cerevisiae</i> and a spontaneous fermentation. Our results showed a higher concentration of anthocyanins and catechins in wines obtained by the sequential inoculation of <i>M. pulcherrima</i> AS3C1 and <i>S. cerevisiae</i>. On the basis of the data obtained, <i>M. pulcherrima</i> AS3C1 possesses an enzymatic profile and some oenological properties that could contribute positively to the definition of the chemical composition of wines, suggesting its possible use for red winemaking processes.