The lipopeptide surfactin produced by certain strains of Bacillus subtillis is a potent biosurfactant with high amphiphilicity and a strong tendency for self-aggregation. Surfactin possesses a number of valuable biological properties such as antiviral, antibacterial, antifungal, and hemolytic activities. Owing to these properties, in addition to the general advantages of biosurfactants over synthetic surfactants, surfactin has potential biotechnological and biomedical applications. Here, the aggregation properties of surfactin in solution together with its behavior at the water/air interface were studied using classical molecular dynamics simulations (MD) at three different pH values. Validation of the MD structural data was performed by comparing neutron reflectivity and volume fraction profiles computed from the simulations with their experimental counterparts. Analysis of the MD trajectories supported conclusions about the distribution, conformations, and interactions of surfactin in solution and at the water–air interface. Considering altogether, the work presented provides atomistic models for the rationalization of some of the structural and dynamic characteristics as well as the modes of action of surfactin at different pH values.