Madeira wine is produced via spontaneous alcoholic fermentation arrested by ethanol addition. The increasing demand of the wine market has led to the need to standardize the winemaking process. This study focuses on identifying the microbiota of indigenous yeasts present during Madeira wine fermentation and then evaluates the impact of selected indigenous non-Saccharomyces as pure starter culture (Hanseniaspora uvarum, Starmerella bacillaris, Pichia terricola, Pichia fermentans, and Pichia kluyveri) in the chemical and phenolic characterization of Madeira wine production. Results showed that the polyphenol content of the wines was influenced by yeast species, with higher levels found in wines produced by Pichia spp. (ranging from 356.85 to 367.68 mg GAE/L in total polyphenols and 50.52 to 51.50 mg/L in total individual polyphenols through HPLC methods). Antioxidant potential was higher in wines produced with Hanseniaspora uvarum (133.60 mg Trolox/L) and Starmerella bacillaris (137.61 mg Trolox/L). Additionally, Starmerella bacillaris stands out due to its sugar consumption during fermentation (the totality of fructose and 43% of glucose) and 15.80 g/L of total organic acids compared to 9.23 g/L (on average) for the other yeasts. This knowledge can be advantageous to standardizing the winemaking process and increasing the bioactive compounds, resulting in the production of high-quality wines.