ABSTRACT Within the human gut microbiota, lactic acid bacteria (LAB) play a crucial role in host health by producing lactic acid, which has been shown to shape microbial interactions and support intestinal homeostasis. However, despite their importance, there are limited insights regarding how LAB species interact with the host and other gut commensals. In this study, the investigation of the human gut microbiota of 10,000 healthy adults allowed the identification of Lactococcus lactis and Streptococcus thermophilus as commonly detected food bacteria. Further in silico analyses led to the identification of reference strains of the L. lactis and S. thermophilus species within the human gut, represented by PRL2024 and PRL2025 strains, respectively, which can represent nomadic bacteria. In vitro experiments revealed that both strains are ecologically adapted to survive and interact within the human gastrointestinal tract, while also highlighting their metabolic capacity to utilize a broad range of carbon sources. Specifically, the lactose metabolism was investigated, revealing that S. thermophilus PRL2025, despite high lactic acid output, incompletely metabolizes galactose, whereas L. lactis PRL2024 ensures full galactose utilization with lower acid production. IMPORTANCE The identification and functional characterization of Lactococcus lactis PRL2024 and Streptococcus thermophilus PRL2025 as human-adapted reference strains provide a valuable foundation for further in vivo experimentation. Given their ecological resilience, metabolic versatility, and interaction potential with beneficial gut microbes, these strains represent promising candidates as microbiota-targeted functional foods.