The benthic foraminiferal assemblages in Lake Varano (southern Italy) have been investigated in detail. Statistical analyses enable us to recognize two main biotopes and five sub-biotopes that reflect different ecological and environmental conditions. The assemblages mainly seem to be influenced by the hydrological (namely salinity) and sediment conditions in the lake. These biotopes are characterized by specific sub-assemblages and variations in relative species’ abundances. The Outer Lake Biotope is affected by marine influence and is dominated by the foraminiferal species Ammonia beccarii, Ammonia parkinsoniana and Aubignyna perlucida, which are more common in open water environments. In contrast, the Inner-Marginal Lake Biotope reflects more restricted conditions where low salinity values and sand are associated with high numbers of miliolids. These two main biotopes are thus subjected to different degrees of confinement and water residence times, both of which are related to the water exchange time of the lake with the Adriatic Sea. In addition five sub-biotopes (Intermediate Marine, Mixing, “Urban”, Marginal, and Innermost) represented by particular foraminiferal assemblages are identified that characterize particular sub-environments. These sub-biotopes are, to some extent, separated by salinity gradients as well as the grain-size and the organic matter of sediments.