Rigidoporus microporus is the main causal of white root disease (WRD) in rubber trees (Hevea brasiliensis). The present study investigates the use of rhamnolipid, a biosurfactant produced by Pseudomonas aeruginosa USM-AR2 against R. microporus. In vitro dose-responses towards rhamnolipid were determined on different isolates of R. microporus using the poisoned food technique (PFT). Inhibition of mycelial growth was found to be dose-dependent, with the highest inhibition of 76.74% at 200 ppm (pH 6.29) on SEG isolate. On the contrary, the lowest concentration of rhamnolipid applied at 10 ppm (pH 5.97) had effectively inhibited the growth of RL 19 to 34.36%. AM isolate was assumed to be the most aggressive pathogen due to the lowest inhibition recorded on all rhamnolipid concentrations tested. At the same time, RL 19 was the least aggressive pathogen compared to the other R. microporus isolates. The rhamnolipid concentrations (ppm), which reduced mycelial growth at 50% (EC50), were recorded at 17.82 ppm for AM isolate, 12.52 ppm for RL 26, and 11.80 ppm for RL 19 isolate. This result indicated that rhamnolipid concentrations to inhibit 50% of mycelial growth might vary based on the aggressiveness and the virulence levels of different R. microporus isolates. It was found that pH changes after incorporating rhamnolipid into the PDA were not the main factor affecting the inhibition of R. microporus isolates. It is obvious that rhamnolipid had an inhibitory effect on fungal growth in vitro. It is the first report on rhamnolipid that has been shown to control R. microporus potentially.