Rigidoporus microporus, which causes white root rot disease (WRD) in Hevea brasiliensis, is a looming threat to rubber plantation in Malaysia. The current study was conducted to determine and evaluate the efficiency of fungal antagonists (Ascomycota) against R. microporus in rubber trees under laboratory and nursery conditions. A total of 35 fungal isolates established from the rubber tree rhizosphere soil were assessed for their antagonism against R. microporus by the dual culture technique. Trichoderma isolates can inhibit the radial growth of R. microporus by 75% or more in the dual culture test. Strains of T. asperellum, T. koningiopsis, T. spirale, and T. reesei were selected to assess the metabolites involved in their antifungal activity. Results indicated that T. asperellum exhibited an inhibitory effect against R. microporus in both volatile and non-volatile metabolite tests. All Trichoderma isolates were then tested for their ability in producing hydrolytic enzymes such as chitinase, cellulase and glucanase, indole acetic acid (IAA), siderophores production, and phosphate solubilization. From the positive results of the biochemical assays, T. asperellum and T. spirale were selected as the biocontrol candidates to be further tested in vivo against R. microporus. The nursery assessments revealed that rubber tree clone RRIM600 pretreated with only T. asperellum or with the combination of T. asperellum and T. spirale was able to reduce the disease severity index (DSI) and exert higher suppression of R. microporus compared to other pretreated samples, with the average DSI below 30%. Collectively, the present study demonstrates that T. asperellum represents a potential biocontrol agent that should be further explored to control R. microporus infection on rubber trees.