Four new cyclodepsipeptides, trichodestruxins A-D (1-4), together with destruxin E2 chlorohydrin (5) and destruxin A2 (6), were isolated from the plant endophytic fungus Trichoderma harzianum by a bioassay-guided fractionation method. Their planar structures were elucidated on the basis of 1D and 2D NMR and MS/MS spectroscopic analyses. The stereochemical configuration was established by application of the advanced Marfey's method, J-based configuration analysis, Mosher's method, and chemical derivatizations. Trichodestruxin B contains hydroxy acid fragments of the THPA unit, while trichodestruxins A and C contain the β-methylproline moiety. All cyclodepsipeptides displayed cytotoxicity against HT-29, A549, and/or P388 cell lines with IC50 values of 0.7-19.1 μM.