Previously phytochemical investigations carried out on the flowers and trunk bark extracts of Citharexylum spinosum L. tree, allowed the isolation of twenty molecules belonging to several families of natural substances [triterpene acids, iridoid glycosides, phenylethanoid glycosides, 8,3'-neolignan glycosides, together with other phenolic compounds]. In the present work, a biological evaluation (anti-tyrosinase, anticholinesterase and cytotoxic activities) was performed on the prepared extracts and the isolated secondary metabolites. The results showed that the EtOAc extract of the trunk bark displayed the highest anti-tyrosinase effect with a percent inhibition of 55.0 +/- 1.8% at a concentration of 100 mu g/mL. The highest anticholinesterase activity was presented by the same extract with an IC50 value of 99.97 +/- 3.01 mu g/mL. The EtOAc extract of flowers and that of the trunk bark displayed the best cytotoxic property with IC50 values of 96.00 +/- 2.85 and 88.75 +/- 2.00 mu g/mL, respectively, against the human cervical cancer cell line (HeLa), and IC50 values of 188.23 +/- 3.88 and 197.00 +/- 4.25 mu g/mL, respectively, against the human lung cancer (A549) cell lines. Biological investigation of the pure compounds showed that the two 8,3'-neolignan glycosides, plucheosides D-1-D-2, generate the highest anti-tyrosinase potency with a percent inhibition of 61.4 +/- 2.0 and 79.5 +/- 2.3%, respectively, at a concentration of 100 mu M. The iridoid glycosides exhibited a significant anticholinesterase activity with IC50 values ranging from 17.19 +/- 1.02 to 52.24 +/- 2.50 mu M. Triterpene pentacyclic acids and iridoid glycosides exerted encouraging cytotoxic effects against HeLa with IC50 values ranging from 9.00 +/- 1.10 to 25.00 +/- 1.00 mu M. The study of the structure-activity relationship (SAR) has been sufficiently and widely discussed. The natural compounds that exhibited the significant bioactivities were docked.