Lactic acid bacteria and bifidobacteria (LAB & Bifido), isolated from the gastrointestinal tract of Apis mellifera intermissa (BGIT), honey (H), propolis (P) and bee bread (BB) of hives set in different vegetations (wildflowers, caraway, orange blossom, Marrubium vulgare, Eucalyptus and Erica cinerea), were subjected to analysis of their antibacterial potential. Isolates able to inhibit Staphylococcus aureus were selected and identified with MALDI-TOF MS leading to 154 strains representing 12 LAB & Bifido species. Lactiplantibacillus plantarum, Pediococcus pentosaceus and Enterococcus faecalis were predominantly found in all matrices. BGIT showed the highest LAB & Bifido diversity with exclusive occurrences of five species (including Bifidobacterium asteroides and Limosilactobacillus fermentum). Honey was the second origin harboring an important variety of LAB species of which Apilactobacillus kunkeei and Enterococcus mundtii were characteristic of both H and BGIT. Principal components analysis revealed associations between antibacterial activities of LAB & Bifido, matrices and honey bee forage plants. Inhibition trends of S. aureus and Citrobacter freundii were highlighted with: Lactiplantibacillus plantarum from BGIT, P, H of bees feeding on Erica cinerea; Pediococcus pentosaceus from BGIT, P, BB associated with Erica cinerea; and Bifidobacterium asteroidesfrom BGIT/orange blossom system. However, Enterococcus faeciumassociated with BGIT/Eucalyptus system antagonized Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Acinetobacter baumannii. Our findings highlighted noteworthy effects of bee forage plants on the antibacterial activity of LAB & Bifido. Our approach could be useful to identify multiple conditions promoting antibacterial potency of LAB & Bifido under the combined effects of feeding plants and living matrices.