Bacteria exhibiting antimicrobial activity were isolated from contaminated sites in Riyadh area, Saudi Arabia. It was charaterized and identified as Rhodococcus erythropolis. The biosurfactants produced by Rhodococcus erythropolis were partially purified and characterized by HPLC and GC-MS. The influence of antimicrobial activities were obtained by using agar diffusion method against some Gram positive and Gram negative pathogenic bacteria involved (Escherichia coli, Pseudomonas aerouginosa, Bacillus cereus, Klebsiella pneumoniae, Proteus spp., Salmonella spp. Corynebacterium spp., Streptococcus pneumoniae and Staphylococcus aureus); and two pathogenic fungi viz., Aspergillus niger and Aspergillus flavus. These surface active agents exhibited high inhibitory activity against Escherichia coli, Pseudomonas aeruginosa, Aspergillus niger and Aspergillus flavus among the tested ones. The results clearly indicated that the antibacterial and antifungal activity of the used glycolipids vary with the species of the organisms used. The microbes examined by scanning electronic microscope (SEM), were totally deformed and exhibited severe destruction. Abnormal cell division was observed at high frequencies among cells that tried to divide in the presence of the Rhodococcus erythropolis glycolipids. Many cells were enlarged, elongated, empty hosts, or fragmented, consistent with the extremely low viability. Thus, the study ascertains the value of the use of these glycolipids which could be of considerable interest to the development of new anti-microbial materials for medical applications as water purification plants, dental surgery equipments and pharmaceutical purposes. The potential activity of these microorganisms to produce useful antimicrobial compounds is great and must be better explored.