Biofilms have a major impact on human health, environment, and industry as they can drive either fundamental ecosystem services or detrimental processes. The type of these biological functions is dependent on the activity, composition and diversity of the microbial communities forming the biofilm. Therefore, natural and/or induced alterations of the community structure by abiotic (for example environmental conditions, shear forces) or biotic (for example predation, grazing, invasion of allochthonous species) factors could have severe consequences on the biofilm functionality. Addition and colonization of genetically modified or unmodified microbial strains were often attempted with the aim of enhancing a desired biological function (for example bioaugmentation). On the contrary, pathogen invasion and survival in beneficial biofilms could be catastrophic, and it is thus trying to be avoided. Nevertheless, the biological interactions between the colonizers (positive or negative) and the existing microbial community in the biofilm are still unclear, though their understanding could have widespread implications and potentials in environmental, industrial and human wellbeing. In this study, we provide insights into the microbial community dynamics of mature biofilms in contact with allochthonous bacterial strains by combining experimental and theoretical approaches. Biofilm growth was initially promoted on polyethylene coupons inserted into bubble column reactors with a volume of 4.6 liters. The reactors were inoculated with activated sludge from a domestic wastewater treatment plant. One bioreactor was used as a control. After 20 days of operation 130 ml of pure microbial suspensions, with known concentration, of Aquabacterium spp., Escherichia coli, Pseudomonas putida, Lactococcus lactis and Leuconostoc mesenteroides were injected to the other reactors over a period of two hours. The capacity of these strains to adhere and colonize the existing biofilm was then surveyed over time and compared against the biofilm in the control unit by quantitative Polymerase Chain Reaction (qPCR). The inclusion of the allochthonous strains added a new competitor for space and resources in the multispecies biofilm, thereby perturbing the naturally occurring microbial community dynamics. The observed biofilm changes in composition and diversity, which were regularly monitored by single-strand conformational polymorphism (SSCP), together with the specific growth kinetics of the allochthonous strain in the biofilm, were then included as assumptions in a conceptual multi-species model. This model will provide a valuable tool for predicting the ecological behavior of a complex biofilm community when colonized by an external species. Consequently, this study will help future manipulation of biofilm diversity for the improvement of a desired biological process and/or the protection of the existing microbial community against adverse pathogens.