Abstract: Background and Objective: Fungal-strains are well familiar to produce potent therapeutic-moieties now days. Trichoderma harzianum is previously used as fungicide and have good applications in the treatment of soil for suppression of pathological microbial-strains. The current study was aimed to produce useful secondary metabolites in maximum quantity by optimizing the required conditions and to evaluate their biological activities. Materials and Methods: The experimental conditions were optimized by using various media (in various volumes), altering the incubation period and temperature, applying uniform and altering pH. Metabolites obtained were investigated for antibacterial, antifungal, cytotoxic, leishmanicidal, phytotoxic, insecticidal, α-chymotrypsin inhibitory and urease inhibitory potential. Results: Results revealed that the growth of T. harzianum was best supported by “Czapek Yeast Broth” at pH 7 with a maximum production of 1840 mg/100 mL followed by Sabouraud Dextrose Broth at pH 7 with 1580 mg/100 mL production with 15 days incubation at 30°C. The T. harzianum showed a significant antibacterial effect (inhibition %) against S. dysentery (96), S. aureus (94), S. typhi (81), E. faecalis (80), A. parasiticus (96), A. flavus (61) and A. niger (50). No leishmanicidal activity was observed in all tested samples. A moderate (65%) phytotoxic and mild (14.6%) insecticidal activity was observed. In enzyme inhibitory assays mild α-chymotrypsin (15.1%) and moderate urease (41.1%) inhibition was observed. Conclusion: The maximum secondary-metabolites of T. harzianum can be obtained by utilizing the above mentioned experimental conditions; that can be best therapeutic alternatives for the treatment of various pathological diseases.