Quinazolinone alkaloids belong to classes of fused heterocycles that are of considerable interest due to a broad spectrum of biological activities and diverse range of pharmacological properties, for example, anticancer, antimicrobial, antifungal, antiviral, antiprotozoan, antidepressant, anti-inflammatory and antimalarial activities. Quinazolinone derivatives are considered an important chemical for the synthesis of various functional units or precursors with physiological significance and pharmacological utility, and their core heterocyclic frameworks are also present in many various drug molecules. Among a diverse group of quinazolinone metabolites from both synthetic and natural origin, 2,4(1H,3H)quinazolinedione motifs are reported to exhibit anticonvulsant activity against electroshock and possess potential antihypertensive properties. Majority of quinazoline-2,4-dione derivatives are known synthetically, whereas the natural occurrence of these molecules is quite uncommon. During our screening program of rare actinomycetes from Thailand in search for novel and biologically active secondary metabolites, investigation of Streptosporangium amethystogenes BCC 27081 led to the isolation of two new compounds, amethysione (1) and amethysamide (2), together with their related derivatives including 2-(3-phenethyl-ureido)-benzoic acid (3), 2-(3-phenethyl-ureido) benzoic acid methyl ester (4), N-phenethyl-2-(3-phenethyl-ureido) benzamide (5), 1,3-diphenethyl-urea (6), 3-(2-phenethyl)-2,4(1H,3H)-quinazolinedione (7), and 1-methyl-3-(2-phenylethyl)-2,4(1H,3H)-quinazolinedione (8). In this paper, we report the isolation, structure elucidation and biological activities of these new metabolites and their derivatives.