The total ethanol extract and its derived ethyl acetate fraction of the soft coral Nephthea mollis displayed remarkable in-vitro anti-trypanosomal potential against Trypanosoma brucei with IC50 value of 6.4 and 3.7 (µg/ml, 72 h respectively. Consequently, the total ethanol extract was subjected to LC-HR-ESI-MS metabolomic profiling to discover the constituents that possibly underlie their bioactivities. Therefore, thirty-three secondary metabolites were characterized, among them, sesquiterpenes and diterpenes were found to prevail. In silico modeling was carried out on the dereplicated compounds to provide an insight into their anti-trypanosomal mechanism of action with docking study on ornithine decarboxylase (ORD) which illustrated that five of the dereplicated compounds (2-deoxy-12-ethoxy-7-O-methyl lemnacarnol, Nephthenol, 4α-O-acetyl-selin-11-en, Eudesma-4,7(11)-diene-8β-ol, and Chabrolidione A) have the highest affinity to the ornithine decarboxylase enzyme. These results highlight the valuable chemical profile of Nephthea mollis as a lead source for anti-trypanosomal natural products.