Different microbial inhibition strategies based on the planktonic bacterial physiology have been known to have limited efficacy on the growth of biofilms communities. This problem can be exacerbated by the emergence of increasingly resistant clinical strains. Biosurfactants have merited renewed interest in both clinical and hygienic sectors due to their potential to disperse microbial biofilms. In this work, we explore the aspects of Bacillus subtilis BBK006 biofilms and examine the contribution of biologically derived surface-active agents (rhamnolipids) to the disruption or inhibition of microbial biofilms produced by Bacillus subtilis BBK006. The ability of mono-rhamnolipids (Rha-C-10-C-10) produced by Pseudomonas aeruginosa ATCC 9027 and the di-rhamnolipids (Rha-Rha-C-14-C-14) produced by Burkholderia thailandensis E264, and phosphate-buffered saline to disrupt biofilm of Bacillus subtilis BBK006 was evaluated. The biofilm produced by Bacillus subtilis BBK006 was more sensitive to the di-rhamnolipids (0.4 g/L) produced by Burkholderia thailandensis than the mono-rhamnolipids (0.4 g/L) produced by Pseudomonas aeruginosa ATCC 9027. Rhamnolipids are biologically produced compounds safe for human use. This makes them ideal candidates for use in new generations of bacterial dispersal agents and useful for use as adjuvants for existing microbial suppression or eradication strategies.