Microorganisms can produce a plethora of secondary metabolites, some acting as signaling compounds, and others as suppressing agents. As yet, the potential of groundwater microbes to produce antimicrobial compounds to increase their competitiveness against other bacteria has not been examined. In this study, we developed an AlamarBlue® based high-throughput screening method that allowed for a fast and highly standardized evaluation of both growth inhibiting and promoting metabolites. With this technique, 149 screened bacterial isolates were grown in monocultures and in 1402 co-cultures. Co-cultivation did not increase the frequency of growth inhibition against the two tested model organisms (S. aureus 533R4 and E. coli WA321) compared to monocultures. Mainly co-cultivation of Proteobacteria induced growth inhibition of both model organisms. Only slightly increased growth promotion of S. aureus 533R4 was observed. Growth promoting effects on E. coli WA321 were observed by supernatants from co-cultures between Bacteroidetes and Firmicutes. With the standardized screening for both, growth inhibiting and promoting effects, this method will enable further studies to elaborate and better understand complex interspecific interactions and networks in aquatic communities as well as in other environments.