Turning lignin from black liquor waste into value-added bioactive agents is one of the possible routes for improving the sustainability and profitability of lignocellulosic industry. However, due to chemical and structural variability of lignin, it is necessary to isolate specific lignin fractions from black liquor with the purpose to achieve samples with unique chemical and structural characteristics and therefore, specific biological activities. In this study, poplar lignin fractions isolated from Kraft black liquor by sequential acid precipitation at pH´s 7.5, 5 and 2.5 (denoted as P-7.5, P-5 and P-2.5) were characterized according to their physicochemical, antioxidant and antibacterial properties. In general, lignin fractions displayed a wide elimination of lateral chains (aryl-β ether and C–C) and, therefore a high phenolic content and low molecular weight, as the pH sequential precipitation was decreased from 7.5 to 2.5. Moreover, thermal analysis revealed that the P-7.5 lignin fraction showed higher thermal stability than P-2.5 and P-5. In terms of antioxidant activity, the P-7.5 lignin fraction, with a higher S/G ratio and a less oxidized structure compared to P-5 and P-2.5, exhibited higher antioxidant activity. In addition, lower antibacterial effect was observed for all lignin fractions against Escherichia coli compared to that obtained against Staphylococcus aureus. Among them, the P-2.5 and P-5 fractions, with higher phenolic content and lower molecular weight values than P-7.5, showed a greater antibacterial effect against S. aureus.