In this work was evaluated the effect of sequential inoculum of Hanseniaspora uvarum AS27 strain and a commercial Saccharomyces cerevisiae yeast on the physical−chemical and organoleptic features of Aglianico, a traditional red wine of Southern Italy. Four fermentation treatments on a pilot scale were performed. In fermentation treatment A, the alcoholic fermentation was spontaneously conducted by the indigenous yeasts present in grape must. In the fermentation treatments B and C were inoculated respectively S. cerevisiae FE and H. uvarum AS27 strains, as a single starter. The fermentation treatment D was initially inoculated with H. uvarum AS27, and S. cerevisiae strain was added after 72 h (sequential inoculation). Microbiological, physical−chemical parameters and sensory profiles of the wines have been defined. The results showed that the use of H. uvarum AS27, in sequential inoculum with S. cerevisiae FE, influenced the wine composition, enriching it in polyphenolic and volatile compounds. Further, the sensory evaluation showed that the use of H. uvarum AS27 strain, in co-culture with S. cerevisiae, gives the wine more pleasant characteristics. Therefore, the results have highlighted how the use of particular non-Saccharomyces yeasts can represent a biotechnological resource in red wine production.