The interstitial groundwater genus Microcharon (Crustacea, Isopoda, Microparasellidae) is highly diversified in southern France. A new species, Microcharon boulanouari n. sp. is described from the Aude River, whereas specimens from the Lachein River in the central Pyrenees are reassigned to another species, M. ariegensis new to Science. Microcharon boulanouari n. sp. is closely related to the species of the group rouchi and may belong to the phylogenetic western Mediterranean lineage. The two-step model of colonization and evolution provides an understanding of the origin and age of this stygobite. Microcharon boulanouari n. sp. is derived from marine ancestors that lived in the interstitial littoral shallow bottoms of the Atlantic embayment which covered southwestern France at the very beginning of the early Eocene period. Both the regression of this gulf at the start of the Eocene and the Pyrenees uplift may have played a major role in the evolutionary history through vicariance of Microcharon boulanouari n. sp. and of the northern Pyrenean species of the group rouchi.