The bio-active materials were isolated from prickly lettuce (Lactuca serriola) by using several extraction solvents. The contents of general compounds, vitamins, polyphenols and flavonoids of prickly lettuce were analysed. In addition, nitrite scavenging activity, anti-oxidative activity and anti-microbial activity of methanol extracts were measured. The ethylacetate (EtOAc) fraction of methanol extracts of prickly lettuce showed the best on the nitrite scavenging activity, anti-oxidant activity and anti-microbial activity. And also, the contents of polyphenols and flavonoids were the highest among all fractions. The subfraction 2 (EtOAc:MeOH=3:1) of EtOAc fraction showed the strongest anti-oxidative activity among 5 subfractions. The subfraction 2 was identified as ρ-coumaric acid and caffeic acid by GC-MS. The anti-microbial activity of each solvent fraction from prickly lettuce was measured. The EtOAc fraction showed the strongest anti-microbial activity. Overall, these results may provide the basic data to understand the biological activities of bio-active materials derived from prickly lettuce.