Cryoconite holes play a significant role in the nutrient cycling on glaciers and can be regarded as a storehouse of nutrients that are generated through microbial and photochemical activities. In this work, the chemical characteristics of hydrologically connected and isolated cryoconite holes from three geographically distinct regions of coastal Antarctica, namely Larsemann Hills, Amery Ice Shelf and central Dronning Maud Land were studied. Major ions (Na+, K+, Mg2+, Ca2+, Cl−, SO42− and NO3−) and total organic carbon in the hydrologically isolated, closed cryoconite holes showed significantly higher enrichment (6–26 times and 9 times, respectively) over the conservative tracer ion Cl− possibly due to sediment dissolution and microbial synthesis during isolation period. In contrast, depletion of major ions and organic carbon were observed in the open, hydrologically connected holes due to their discharge from the cryoconite holes through interconnected streams. This study suggests that the contribution of cryoconite holes to the nutrient and microbial transport to downstream environments may vary with the extent of hydrological connectivity by virtue of the fact that nutrients and organic carbon which accumulate in the isolated cryoconite holes during isolation could get washed to downstream environments in the event that they get connected through surface or subsurface melt channels.