Nettle is a widely known plant whose high biological activity and beneficial medicinal effects are attributed to various bioactive compounds, among which polyphenols play an important role. In order to isolate polyphenols and preserve their properties, advanced extraction techniques have been applied to overcome the drawbacks of conventional ones. Therefore, microwave-assisted extraction (MAE) has been optimized for the isolation of nettle leaves polyphenols and it was compared to pressurized liquid extraction (PLE) and conventional heat-reflux extraction (CE). The obtained extracts were analyzed for their individual phenolic profile by UPLC MS and for their antioxidant capacity by ORAC assay. MAE proved to be the more specific technique for the isolation of individual phenolic compounds, while PLE produced extracts with higher amount of total phenols and higher antioxidant capacity. Both techniques were more effective compared to CE. PLE nettle extract showed antimicrobial activity against bacteria, especially against Gram-negative ATCC 4973 and NCTC 11168 strains. This suggests that PLE is suitable for obtaining a nettle extract with antioxidant and antimicrobial potential, which as such has great potential for use as a value-added ingredient in the food and pharmaceutical industry.