Erismadelphus exsul Mildbr bark is widely used in Gabonese folk medicine. However, little is known about the active compounds associated with its biological activities. In the present study, phytochemical profiling of the ethanolic extract of Erismadelphus exsul was performed using a de-replication strategy by coupling HPLC-ESI-Q/TOF with a molecular network approach. Eight families of natural compounds were putatively identified, including cyclopeptide alkaloids, esterified amino acids, isoflavonoid- and flavonoid-type polyphenols, glycerophospholipids, steroids and their derivatives, and quinoline alkaloids. All these compounds were identified for the first time in this plant. The use of molecular networking obtained a detailed phytochemical overview of this species. Furthermore, antioxidant (2,2-diphenyl-1-picryl-hydrazylhydrate (DPPH) and ferric reducing capacity (FRAP)) and in vitro antimicrobial activities were assessed. The crude extract, as well as fractions obtained from Erismadelphus exsul, showed a better reactivity to FRAP than DPPH. The fractions were two to four times more antioxidant than ascorbic acid while reacting to FRAP, and there was two to nine times less antioxidant than this reference while reacting to DPPH. In addition, several fractions and the crude extract exhibited a significant anti-oomycete activity towards the Solanaceae phytopathogen Phytophthora infestans in vitro, and, at a lower extent, the antifungal activity against the wheat pathogen Zymoseptoria tritici had growth inhibition rates ranging from 0 to 100%, depending on the tested concentration. This study provides new insights into the phytochemical characterization and the bioactivities of ethanolic extract from Erismadelphus exsul bark.