Biosurfactants production is a great advantage for those microorganisms that could be involved in bioremediation processes. In the present study, 164 bacteria and 212 fungi isolated from a contaminated site were investigated for their capability to produce anionic biosurfactants. The screening was carried out by the blue agar test, providing semi-quantitative results for more than 370 strains. Bacteria response was easy to evaluate whereas fungi showed a plethora of responses that partially interfere with the evaluation of positive results. Among the tested strains, 43 bacteria and 20 fungi were identified as good producers of anionic biosurfactants, as indicated by the formation of a clear dark blue halo around the colony.