Trichoderma harzianum is a fungus that are important in the trade as a biocontrol. However, the identified species and the structure of the active substance which played a role in biocontrol is not clear. So, in this study the antimicrobial activity and biochemical properties of crude cell extracted and culture supernatant from Trichoderma harzianum strain T9 were analyzed. The results showed that both of crude cell extracted and culture supernatant are able to inhibit growth of 10 microorganisms. Furthermore, SDS-PAGE was used to study the protein expression cell from both samples. The result showed many protein bands from both sources were detected. However, the SDS-PAGE displayed high protein concentration from culture supernatant. Both culture supernatant and crude cell extracted showed heat-stable antimicrobial activity after sterilization at 100 0C for 15 minutes and 121 0C for 15 min, respectively. Both of them are also resist to pronase digestion. These results indicate that the antibacterial substance from Trichoderma harzianum isolate T9 might not be a protein in nature.