The combination of micro-segmented flow with miniaturized flow-through multisensor-technology has been utilized for metabolite profiling of soil bacteria. Series of sub-μl segments were generated containing soil sample slurry from historic copper mining sites and exposed to heavy metal salts of copper and nickel. Segments were examined for bacterial growth and spectral properties as well as for the effect of heavy metal-treatment after different incubation times. In order to evaluate microbial growth, extinction was recorded with 4 different spectral channels. Fluorescence was measured using a microflow-through fluorometer to detect both growth and production of fluorescent dyes or metabolites. The incidence of single segments with enhanced absorption in one of the spectral channels or enhanced fluorescence was scored to detect soil microorganisms with interesting properties for further screening. The study could show that the number of vegetated segments, the density of microorganisms in the segments after cultivation and the spectral response are different for separate soil samples and different metals. Thus, the highly parallelized and miniaturized segmented flow method is a promising tool for profiling of soil samples with regard to identifying micro-organisms with interesting profiles for secondary metabolite-production.