The winery industry generates significant amounts of organic waste, such as pruning firewood, stems, and wine lees, which can cause environmental issues and affect the economic sustainability and competitiveness of the industry. Given the known antioxidant and antimicrobial properties of phenolic compounds, we analyzed these by-products to quantify their phenolic composition and evaluate the effectiveness of pruning firewood and stem extracts from six autochthonous varieties of the Dão Region in inhibiting the growth of bacteria from diabetic foot wounds isolated from hospital patients. The study employed colorimetric methods to measure total phenols, ortho-diphenols, and flavonoids in the phenolic composition. The ABTS, DPPH, and FRAP methods were applied to assess the antioxidant capacity, and the disk diffusion method was applied to determine the antimicrobial activity of Gram-positive and Gram-negative bacteria. The results showed that Jaen had the most ortho-diphenols and flavonoids in pruning firewood, and the highest levels of these compounds were located in stem extracts. Jaen also had the highest antioxidant capacity in both pruning firewood and stems across all methods used. Notably, red wine lees displayed the highest biological and antioxidant activities. Moreover, pruning firewood extract displayed great efficacy in inhibiting the growth of Gram-positive bacteria, making it a promising candidate as a natural alternative against antibiotic resistance, which is a global public health concern.