This is the report of the HPLC-MSn-guided isolation of new anti-inflammatory prenylated isoflavonoids and pterocarpans from the stems of Acosmium diffusissimum using GNPS molecular networking as the main tool. Cluster analysis guided the isolation of five new prenylated isoflavonoids, diffusiflavone A-E (1-4 and 9), two prenylated pterocarpans, diffusicarpan A and B (5 and 6), and two known compounds 6-prenylorobol (7) and 3-O-methylquercetin (8). The in vitro anti-inflammatory potential of compounds 1-6 and 9 was assessed in macrophages induced with lipopolysaccharide (LPS). Compounds 2, 3, 5, and 9 were observed to have a reduction in NO levels in at least one of the concentrations tested (1.25, 2.5, 5, 10, and 20 μg/mL) and also reduced IL-1β and IL-6 cytokines, especially diffusicarpan A which reduced cytokine levels in all the concentrations tested.