Heavy crude oils are comprised of different fractions of asphaltenes, resins, aromatic and saturated compounds, which contribute to their high viscosities, making them intrinsically difficult to biodegrade. Besides, contaminated soils that contain limestone rocks are preferably oil wet, which makes their removal difficult. Biosurfactants are capable of modifying the wettability of rock/oil/water system and reduce interfacial tension, which promotes the release of oil, making it bioavailable. The objective of this work was to produce and evaluate a biosurfactant biosynthesized by a strain of Bacillus subtilis to improve the biodegradation of a heavy crude oil impregnated in limestone sand. The biosurfactant production was carried out in a 3L stirred reactor. The biosurfactant activity was determined at different temperatures, pH and salinities to establish its stability. Interfacial tension and contact angle were evaluated to determine the mechanisms involved in heavy oil release. Finally, the effect of the biosurfactant on the release of heavy oil impregnated in limestone sand was elucidated, for its subsequent biodegradation using a strain of Rhodococcus sp. It was found that the maximum CO₂ production rate, used as an indicator of biological activity, improved by 32.84% in systems where biosurfactant was used, compared to those where it was not used. At least 67% of the fraction > C₂₀ was degraded in the systems with biosurfactant, reducing the aromatic and resin fractions (12.5% and 2%, respectively), while in systems without biosurfactant only 37% was removed of the fraction > C₂₀ and the reduction of aromatics and resins was null. The biosurfactant proved to be effective in the release of crude oil and significantly improved the biodegradation process.