In this work, the antifungal activity of rhamnolipids produced by Pseudomonas aeruginosa #112 was evaluated against Aspergillus niger MUM 92.13 and Aspergillus carbonarius MUM 05.18. It was demonstrated that the di-rhamnolipid congeners were responsible for the antifungal activity exhibited by the crude rhamnolipid mixture, whereas mono-rhamnolipids showed a weak inhibitory activity. Furthermore, in the presence of NaCl (from 375 mM to 875 mM), the antifungal activity of the crude rhamnolipid mixture and the purified di-rhamnolipids was considerably increased. Dynamic Light Scattering studies showed that the size of the structures formed by the rhamnolipids increased as the NaCl concentration increased, being this effect more pronounced in the case of di-rhamnolipids. These results were confirmed by Confocal Scanning Laser Microscopy, which revealed the formation of giant vesicle-like structures (in the µm range) by self-assembling of the crude rhamnolipid mixture in the presence of 875 mM NaCl. In the case of the purified mono- and di-rhamnolipids, spherical structures (also in the µm range) were observed at the same conditions. The results herein obtained demonstrated a direct relationship between the rhamnolipids antifungal activity and their aggregation behaviour, opening the possibility to improve their biological activities for application in different fields.