Rock dwelling organisms (lithobionts) such as cyanobacteria (prokaryotes) and chlorolichens (eukaryotes) abound in the Negev Desert, where they cover almost all calcareous bedrocks and rock particles (cobbles, boulders). In a small limestone watershed in the Negev Highlands, cyanobacteria inhabit the south-facing (SF) bedrocks, epilithic lichens (accompanied by endolithic lichens) inhabit the north-facing (NF) bedrocks, while endolithic lichens cover most of the cobbles and boulders in both aspects. In order to study their contribution to runoff water, a pair of runoff plots was established on habitats with cyanobacteria, endolithic lichens, and epilithic lichens. Rain and runoff were collected during the hydrological year 2006/07, and the chemical composition (Ca, Mg, Na, K, Cl, SO4, HCO3, Si) of the rain and runoff water was analyzed. Several patterns were observed: (a) as indicated by Si, more dust accumulated on the bedrocks; (b) all substrates exhibited high amounts of Ca, and HCO3; (c) while SF-bedrocks showed enrichment in K, both bedrocks (and especially the NF bedrocks), as well as the NF boulders showed an enrichment in Mg. While the enrichment in Ca and HCO3 can be explained by the contribution of the limestone parent material, the enrichment in K and Mg can be explained by the contribution of the living lithobionts, with K being mainly contributed by the cyanobacteria and Mg mainly by the epilithic lichens. Ion enrichment may therefore be aspect-dependent, reflecting the lithobiont distribution within the drainage basin, partially explaining the enrichment in K and Mg previously recorded in runoff water from the Negev.