Trichoderma species are known to produce the molecules with promising anticancer, antimicrobial, and antiviral activities. Likewise, we previously reported a metabolite (TM2) from T. atroviride as an anti-prostate cancer agent but its activity was limited due to less ability to target the cancer cells. Therefore, the present work aimed to load the different concentrations of TM2 (1.25-20 mg.mL(-1)) in chitosan nanoparticles (TM2-CNPs) to improve its cytotoxicity in PC3 cells. The input of 10 mg.mL(-1) of TM2 was achived spherical shaped TM2-CNPs with good drug entrapment efficiency of 50.97 +/- 0.12% and drug loading capacity of 24.68 +/- 0.64%. The TM2-CNPs exhibited an average size of 187.8 +/- 1.51 nm, PDI of 0.197 +/- 0.02, and zeta potential of 21.4 +/- 0.17 mV. The FTIR spectra showed the functional group (NH2 and O H) of chitosan in TM2-CNPs. The pH-dependent release was found 50% in the pH 5.8 while < 10% in pH 7.4. The IC50 dosage of TM2-CNPs treatment was caused 60.48% cell death in the PC3 cells through activation of caspase 3 and inhibition of the BCL-2 expression. Overall, this work has demonstrated that the pH-sensitive release of TM2 from nanocarrier (CNPs) increased anti prostate cancer activity.