Abstract: The antimicrobial substances of Antarctic psychrotrophic bacterium Pseudomonas sp. P4-11 were studied in this paper. The results of study on temperature and pH stability of bioactive substances of fermented culture showed that it had better stability at the range of 30~60 and pH 6~10. By bioassay-guided, two antimicrobial substances (T2-20-7-11-5 and T2-20-7-11-7) were isolated by ethyl acetate extraction, silica gel column chromatograph and HPLC. The structure of T2-20-7-11-5 and T2-20-7-11-7 were both identified as nonylphenol ethoxylates. The effect of T2-20-7-11-5 on the ultrastructure of Fusarium oxysporum spore was observed by scanning electron microscope (SEM). The compound might have a major role in the cell wall or cell membrane, causing leakage of cell contents and consequently resulting in spores death.