Volatile organic compounds (VOCs) produced by microorganisms are known both for their effect on pathogens and their role as mediators in various interactions and communications. Previous studies have demonstrated the importance of VOCs for ecosystem functioning as well as their biotechnological potential, but screening for bioactive volatiles remained difficult. We have developed an efficient testing assay that is based on two multi-well plates, separated by a sealing silicone membrane, two tightening clamps, and variable growth media, or indicators. The experiment design as presented here is a novel and robust technique to identify positive as well as negative VOC effects on the growth of a target organism and to test for specific substances e.g., hydrogen cyanide which can be detected with a suitable indicator. While the first pre-screening assay is primarily based on indicator color change and visible growth diameter reduction, we also introduce an advanced and quantitatively precise experiment design. This adaptation involves qPCR-based quantification of viable target cells after concluding the treatment with VOCs. Therefore, we chose preselected active isolates and compared the partial 16S rRNA gene copy number of headspace-exposed E. coli with non-treated controls. Separately obtained headspace SPME and GC/MS-based profiles of selected bacterial isolates revealed the presence of specific and unique signatures which suggests divergent modes of action. The assay was evaluated by screening 100 isolates of lung lichen-associated bacteria. Approximately one quarter of the isolates showed VOC-based antibacterial and/or antifungal activity; mainly Pseudomonas and Stenotrophomonas species were identified as producers of bioactive volatiles.