The goal of this study was to assess the presence and surfactant potential of naturally occurring microbes from a tropical soil with petrochemical contamination. Microorganisms in a soil sample from a Brazilian landfarm were isolated and grown on petroleum as the sole carbon source. Of 60 isolates screened for petroleum-based growth, 7 demonstrated surfactant activities by the drop-collapse methodology over various types of oils. From their growth profiles in liquid culture during 132 h, all had their first detection of surfactant activity after 96 h. Little is currently known about biosurfactant-producing microorganisms in tropical environments contaminated by hydrophobic compounds, and the search for them is essential for bioremediation and for oil recovery enhanced by microbes. Our results indicate that different petroleum-grown microorganisms showing surfactant activity can be recovered from landfarm soil in a tropical environment.