The secondary metabolites from the cultured supernatant of Serratia ureilytica TKU013 with squid pen as the sole carbon/nitrogen source were isolated and ascertained the mechanism of biological activity. The EtOAc layer, which has high DPPH scavenging activity, was applied to silica gel column chromatography with a gradient of CH(2)Cl(2)/MeOH solvent system, to yield A-H and MeOH fractions. The DPPH scavenging activity and cytotoxic activities against Doay and HEp-2 cell lines of these fractions were examined. The active fractions were further applied to purification by RP-HPLC, to obtain seven compounds, including a novel compound, serlyticin-A (1), together with six known compounds, serranticin (2), serratamolide A (3), thymine (4), (4-hydroxyphenyl)acetic acid (5), methyl p-hydroxybenzoate (6), and uracil (7). Their structures were determined by physical and extensive spectral analyses such as 1D and 2D NMR data, as well as comparison with literature values. Furthermore, the major secondary metabolites of EtOAc extract of the cultured supernatant were examined by the fingerprinting data of the HPLC system.