Abstract Bacteroidota, a diverse phylum of bacteria, includes classes whose members are increasingly recognized for their significant contributions to host health, particularly through their antimicrobial properties. This study investigates the functional diversity of 42 new Bacteroidia and Sphingobacteriia strains enriched and identified from diverse hosts, including mouse ceca and human stool samples. Using 16S rRNA gene sequencing, we phylogenetically characterized the strains of the genera Bacteroides, Phocaeicola, and Sphingobacterium and assessed their functional properties related to potential beneficial functions. The strains were evaluated concerning their ability to inhibit biofilm formation of the World Health Organization-declared clinically significant pathogens, including Gram-positive Staphylococcus aureus and Staphylococcus epidermidis, Gram-negative Klebsiella oxytoca and Pseudomonas aeruginosa, and the eukaryotic yeast Candida albicans. Additionally, we investigated bile salt hydrolase and quorum-quenching (QQ) activities of the strains, as these functions contribute to microbial community interactions and host–microbe dynamics. Our findings demonstrate that all examined Bacteroidota strains consistently exhibit a capacity to inhibit biofilm formation but to different extents. Furthermore, 14 strains showed QQ activity, and 39 bile salt hydrolase activity, indicating functional diversity among the isolates. High biofilm inhibition as well as QQ activity against both autoinducers, AHL and AI-2, were predominantly observed in Bacteroides caecimuris and Bacteroides muris. These traits suggest that such strains may play important roles in shaping microbial communities and interfering with pathogens and their communication. Overall, this study provides valuable insights into strain-specific functions that could support future microbiome-based strategies for pathogen control and host health modulation.