ABSTRACTAs common commensals residing on mucosal tissues,Lactobacillusspecies are known to promote health, while someStreptococcusspecies act to enhance the pathogenicity of other organisms in those environments. In this study we used a combination ofin vitroimaging of live biofilms and computational modeling to explore biofilm interactions betweenStreptococcus oralis, an accessory pathogen in oral candidiasis, andLactobacillus paracasei, an organism with known probiotic properties. A computational agent-based model was created where the two species interact only by competing for space, oxygen, and glucose. Quantification of bacterial growth in live biofilms indicated thatS. oralisbiomass and cell numbers were much lower than predicted by the model. Two subsequent models were then created to examine more complex interactions between these species, one whereL. paracaseisecretes a surfactant and another whereL. paracaseisecretes an inhibitor ofS. oralisgrowth. We observed that the growth ofS. oraliscould be affected by both mechanisms. Further biofilm experiments support the hypothesis thatL. paracaseimay secrete an inhibitor ofS. oralisgrowth, although they do not exclude that a surfactant could also be involved. This contribution shows how agent-based modeling and experiments can be used in synergy to address multiple-species biofilm interactions, with important roles in mucosal health and disease.IMPORTANCEWe previously discovered a role of the oral commensalStreptococcus oralisas an accessory pathogen.S. oralisincreases the virulence ofCandida albicansinfections in murine oral candidiasis and epithelial cell models through mechanisms which promote the formation of tissue-damaging biofilms.Lactobacillusspecies have known inhibitory effects on biofilm formation of many microbes, includingStreptococcusspecies. Agent-based modeling has great advantages as a means of exploring multifaceted relationships between organisms in complex environments such as biofilms. Here, we used an iterative collaborative process between experimentation and modeling to reveal aspects of the mostly unexplored relationship betweenS. oralisandL. paracaseiin biofilm growth. The inhibitory nature ofL. paracaseionS. oralisin biofilms may be exploited as a means of preventing or alleviating mucosal fungal infections.