Coffee Wilt Disease (CWD) is a vascular disease caused by the fungal pathogen; Fusariumrnxylarioides and one of the most important diseases of coffee that was prevalent in Ethiopia. Thernuse of indigenous antagonistic isolates of Trichoderma would be a nature conserving means torncombat this disease. The current research work was designed to isolate, extract, characterize,rnevaluate and determine the antifungal compounds from Trichoderma isolates against F.rnxylarioides. An experiment was conducted to extract, characterize, purify, evaluate and determinernthe antagonistic potential of antifungal compounds from six Trichoderma isolates that wererninhibitory towards F. xylarioides. For extraction of antifungal compounds from fungal myceliumrnor culture media different organic solvents: CHCl3, EtOH, MeOH, EtOAc, n-hexane, and butanernwere used. The chloroform, ethanol and butane extracts were screened for their antifungalrnactivity. A direct bioautographic procedure, involving spraying suspension of F. xylarioides onrnTLC plates developed in solvents of varying polarities was used to detect a number of antifungalrncompounds present in the extracts. Moreover, in-vitro antagonistic bioassays were performed torntest, evaluate and determine the potentiality of Trichoderma isolates as biological control agentsrnagainst F. xylarioides. The study indicated that antifungal compounds were successfully extractedrnfrom fungal culture media with all organic solvents used except hexane. For purification andrnseparation of crude extracts on a TLC, the optimum Rf value was obtained by only three extractingrnsolvents: CHCl3, EtOH and Butane using seven pre-screened solvent systems. Bioautographyrnassay revealed 60 zones of inhibition spots and the highest inhibition zone was observed in AUT5rn(51 mm) and AUT6 (44 mm) with EtOH extract at Rf value of 0.43. In in-vitro bioassay, the highestrnmean inhibitory effect on the growth of the pathogen was achieved by AUT2 (77.4%) isolaternfollowed AUT3 (72.9) in dual culture. The minimum inhibitory concentration (MIC) of non-volatilerncompounds was observed in isolate AUT3 and AUT6 to be 5% of culture filtrate. In general, TLCdirectedrnbioautography assay was useful in isolating active compounds with antifungal activityrnand all Trichoderma isolates were accounted for reduction in mycelial growth of the test pathogenrnin-vitro.rnrnKeywords: Antagonistic activity, Bioassay methods, Bioautography, Organic solvent polarity, Thinrnlayer chromatography