To survive within complex environmental niches, including the human host, bacteria have evolved intricate inter-species communities driven by competition for limited nutrients, cooperation via complementary metabolic proficiencies, and establishment of homeostatic relationships with the host immune system. Such complex, interdependent relationships have hampered attempts to culture many bacterial strains in research settings, where standard readout of co-culture experiments are usually limited to the relative abundance of each species. Here, we utilize a microfluidic-based co-culture system to characterize dynamic interactions between multiple oral bacterial isolates. Using time-lapse imaging, we define species-specific effects on spatial community relationships during co-culture of Streptococcus species and Staphylococcus aureus with Actinomyces species. Co-culture of Streptococcus cristatus or S. salivarius in nanoliter compartments with Actinomyces graevenitzii revealed localized exclusion of Streptococcus and Staphylococcus from media immediately surrounding A. graevenitzii micro colonies. This community structure did not occur with S. mitis or S. oralis strains, or in co-cultures containing other Actinomycetaceae species such as S. odontolyticus or A. naeslundii. Moreover, fewer neutrophils were attracted to compartments containing both A. graevenitzii and Staphylococcus aureus than to equal number of either species alone, suggesting a possible survival benefit from the interaction.