Microbial community behavior is coupled to a set of genetically-regulated chemical signals that correlate with cell density-the quorum sensing (QS) system-and there is growing appreciation that the QS-regulated behavior of bacteria is chemically, spatially, and temporally complex. In addition, while it has been known for some time that different species use different QS networks, we are beginning to appreciate that different strains of the same bacterial species also differ in their QS networks. Here, we combine mass spectrometric imaging (MSI) and confocal Raman microscopy (CRM) approaches to investigate co-cultures involving different strains (FRD1 and PAO1C) of the same species (Pseudomonas aeruginosa) as well as those involving different species (P. aeruginosa and E. coli). Combining MSI and CRM makes it possible to supersede the limits imposed by individual imaging approaches and enables the spatial mapping of individual bacterial species and their microbial products within a mixed bacterial community growing in situ on surfaces. MSI is used to delineate the secretion of a specific rhamnolipid surfactant as well as alkyl quinolone (AQ) messengers between FRD1 and PAO1C strains of P. aeruginosa, showing that the spatial distribution and production of AQ messengers in PAO1C differs substantially from that of FRD1. In the case of multiple species, CRM is used to show that the prolific secretion of AQs by the PAO1C strain of P. aeruginosa is used to mediate its interaction with cocultured E. coli.