Microbial growth confinement using liquid scaffolds based on an aqueous two-phase system (ATPS) is a promising technique to overcome the challenges in microbial–mammalian co-culture in vitro. To better understand the potential use of the ATPS in studying these complex interactions, the goal of this research was to characterize the effects of bacteria loading and biofilm maturation on the stability of a polyethylene glycol (PEG) and dextran (DEX) ATPS. Two ATPS formulations, consisting of 5% PEG/5% DEX and 10% PEG/10% DEX (w/v), were prepared. To test the containment limits of each ATPS formulation, Escherichia coli MG1655 overnight cultures were resuspended in DEX at optical densities (ODs) of 1, 0.3, 0.1, 0.03, and 0.01. Established E. coli colonies initially seeded at lower densities were contained within the DEX phase to a greater extent than E. coli colonies initially seeded at higher densities. Furthermore, the 10% PEG/10% DEX formulation demonstrated longer containment time of E. coli compared to the 5% PEG/5% DEX formulation. E. coli growth dynamics within the ATPS were found to be affected by the initial bacterial density, where colonies of lower initial seeding densities demonstrate more dynamic growth trends compared to colonies of higher initial seeding densities. However, the addition of DEX to the existing ATPS during the growth phase of the bacterial colony does not appear to disrupt the growth inertia of E. coli. We also observed that microbial growth can disrupt ATPS stability below the physical carrying capacity of the DEX droplets. In both E. coli and Streptococcus mutans UA159 colonies, the ATPS interfacial tensions are reduced, as suggested by the loss of fluorescein isothiocyanate (FITC)–DEX confinement and contact angel measurements, while the microbial colony remained well defined. In general, we observed that the stability of the ATPS microbial colony is proportional to polymer concentrations and inversely proportional to seeding density and culture time. These parameters can be combined as part of a toolset to control microbial growth in a heterotypic co-culture platform and should be considered in future work involving mammalian–microbial cell interactions.