Red rot disease is the major constraint for sugarcane production in India and the pathogen has gained virulence in recent years. About 33 % reduction in yield was observed and loss in sucrose and commercial cane sugar was estimated upto 32 to 50 % in average infections. The present investigation was carried out in sugarcane breeding institute, Coimbatore to study the effect of Psoralea corylifolia hairy root extract against high intensity Colletotrichum falcatum spore suspension (106 spores ml-1) causing red rot disease reaction in canes under Controlled Condition Treatment (CCT) Chamber. Nodal infection, green top, internodal discoloration and internal discoloration of the canes in CCT chamber were taken as the parameters for fixing the disease evaluation after 10 days of incubation. The results of CCT method authenticated the results obtained under laboratory conditions. The study revealed 100 per cent effectiveness of two per cent P. corylifolia hairy root extract over red rot pathogen infection when compared to canes treated only with spore suspension of C. falcatum. Article Citation: Rajkumar D and Murugesan R. Antifungal Activity of Psoralea corylifolia Hairy Root Extract against Sugarcane Red Rot Pathogen under Controlled Condition Treatment Chamber. Journal of Research in Agriculture (2013) 2(2): 173-179. Full Text: http://www.jagri.info/documents/AG0047.pdf