Hypothesizing that rock-dwelling (lithobionts) or soil (loess)-dwelling biocrusts may shed light on the phenomena, we conducted sprinkling experiments in the Negev Highlands. Sprinkling was conducted on 4 types of lithobionts: cyanobacteria which inhabit the south-facing bedrock (ENC), epilithic lichens, inhabiting the inclined (EPIi) and the flat (EPIf) north-facing bedrocks, and endolithic lichens (ENL) inhabiting south-facing boulders. Additional sprinkling took place on two types of soil biocrusts, a mixed crust composed of cyanobacteria, lichens and mosses at the north-facing footslope and a cyanobacterial crust at the more xeric south-facing footslope. The runoff water (of 5 and for 4 plots for each lithobiont and soil biocrust type, respectively) was analyzed for the ionic composition of Na+, K+, Mg2+, Ca2+, NH4+, Cl−, SO42−, and NO3−, whereas HCO3− was calculated. In comparison to rainwater, all habitats (except for K+ in ENL) showed high enrichment ratios (ERs) in K+ and Mg2+, which, unlike the high ERs of the other ions (such as SO42− that may stem from gypsum dissolution), could not have been explained by the rock lithology, clay or dust composition. It is suggested that following wetting, K+, serving for osmoregulation of cells, is released by the crust organisms, being thus responsible for K+ enrichment, while chlorophyll degradation is responsible for Mg2+ enrichment. It is suggested that rock- and soil-dwelling microorganisms may explain K+ and Mg2+ enrichment in runoff and floodwater and subsequently in groundwater of the Negev, and possibly in other arid zones worldwide, affecting in turn the quality of irrigation and drinking water.