Microorganisms have evolved complex systems to respond to environmental signals. Gradients of particular molecules and elemental ions alter the behavior of microbes and their distribution within their environment. Microdevices coupled with automated image-based methods are now employed to analyze the instantaneous distribution and motion behaviors of microbial species in controlled environments at small temporal scales, mimicking, to some extent, macro conditions. Such technologies have so far been adopted for investigations mainly on individual species. Similar versatile approaches must now be developed for the characterization of multiple and complex interactions between a microbial community and its environment. Here, we provide a comprehensive step-by-step method for the characterization of species-specific behavior in a synthetic mixed microbial suspension in response to an environmental driver. By coupling accessible microfluidic devices with automated image analysis approaches, we evaluated the behavioral response of three morphologically different telluric species (Phytophthora parasitica, Vorticella microstoma, Enterobacter aerogenes) to a potassium gradient driver. Using the TrackMate plug-in algorithm, we performed morphometric and then motion analyses to characterize the response of each microbial species to the driver. Such an approach enabled to confirm the different morphological features of the three species and simultaneously characterize their specific motion in reaction to the driver and their co-interaction dynamics. By increasing the complexity of suspensions, this approach could be integrated in a framework for phenotypic analysis in microbial ecology research, helping to characterize how key drivers influence microbiota assembly at microbiota host-environment interfaces.