Pyroligneous acid (PA) obtained from slow pyrolysis of palm kernel shell (PKS) has high total phenolic contents and exhibits various biological activities including antioxidant, antibacterial and antifungal. In this study, PA obtained using slow pyrolysis method and fractionated using column chromatography was characterized (chemical and antioxidative properties) and investigated for its cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LOX) inhibition activities using the in vitro and in silico approaches. The F9 PA fraction exhibited highest total phenolic content of 181.75 ± 17.0 μg/mL. Fraction F21-25 showed ferric reducing antioxidant power (FRAP) (331.80 ± 4.60 mg TE/g) and IC50 of 18.56 ± 0.01 μg/mL towards COX-2 and 5.25 ± 0.03 μg/mL towards the 5-LOX enzymes, respectively. Molecular docking analysis suggested favourable binding energy for all chemical compounds present in fraction F21-25, notably 1-(2,4,6-trihydroxyphenyl)-2-pentanone, towards both COX-2 (- 6.9 kcal/mol) and 5-LOX (- 6.4 kcal/mol) enzymes. As a conclusion, PA from PKS has the potential to be used as an alternative antioxidant and antiinflammatory agents which is biodegradable and a more sustainable supply of raw materials.