Psoriasis is a dermatological inflammatory disease treated for centuries with pyrolytic pine bio-oils (pine tars), although their chemical composition and mechanism of action are poorly investigated. This study focuses on the fractionation of a standardized pine pyrolysis bio-oil, its chemical characterization (GC–MS, UV fluorescence, ESI-MS, and NMR), and the analysis of the anti-IL17 activity of fractions on HaCaT keratinocytes. Furthermore, the impact of the fraction showing the highest anti-IL-17 activity on keratinocyte gene expression was determined. Pine bio-oil comprises molecules derived from cellulose, hemicellulose, and lignin thermochemical degradation reactions, such as hydroxyacetaldehyde, levoglucosan, acetic acid, and phenols. The aqueous fraction exhibited the most consistent anti-IL-17 activity. The functional analysis suggested that this fraction affected the expression of 29 genes associated with the negative regulation of the mitogen-activated protein kinase (MAPK) activity, a pathway known for its crucial role in the inflammatory immune response.