Lactobacillaceae are Gram-positive, and lactic acid-positive (LAB) bacteria frequently serve as probiotics. We first systematically compared five LAB strains for the effects of different carbohydrates on their free-living and biofilm lifestyles. We found that fermentable sugars triggered an altered carrying capacity with strain specificity during planktonic growth, calling for adding a buffering system during the formulation of probiotics. In addition, heterogeneous response to fermentable sugars was manifested in microbial aggregation (measured by image-stream flow cytometry), colony development, and attachment to mucin. Of all probiotic strains, L. rhamnosus GG (LGG), a prevalent probiotic specie, manifested an enhanced survival of self-imposed acid stress, consistent with enhanced cell wall modulation observed by transmitting electron microscopy and proteomic analysis. A comprehensive proteomic and metabolomic study revealed that the formation of biofilms and aggregation capacity is a specific response to glucose and independent of self-imposed acid stress. In contrast, the increased competitiveness and aggression of LGG and other LAB strains towards enteric pathogens were a synergistic outcome of a change in organic acid production, glucose-dependent bacteriocin production, and fermentation-specific volatile production. Our improved resolution into the cellular circuits (metabolome, proteome, and volatilome) of probiotic strains and their interactions can lead to developing novel therapeutic approaches to combat GI tract infections.