Streptococcus agalactiae (S. agalactiae), group B Streptococcus (GBS), a major cause of infection in a wide variety of diseases, have been compared in diferent human and animal sources. We aimed to compare the bacterial proteome and metabolome profles of human and animal S. agalactiae strains to delineate biological interactions relevant to infection. With the innovative advancement in mass spectrometry, a comparative result between both strains provided a solid impression of diferent responses to the host. For instance, stress-related proteins (Asp23/Gls24 family envelope stress response protein and heat shock protein 70), which play a role in the survival of GBS under extreme environmental conditions or during treatment, are highly expressed in human and animal strains. One human strain contains ꞵ-lactamase (serine hydrolase) and bioflm regulatory protein (lytR), which are important virulence regulators and potential targets for the design of novel antimicrobials. Another human strain contains the aminoglycosides-resistance bifunctional AAC/APH (A0A0U2QMQ5) protein, which confers resistance to almost all clinically used aminoglycosides. Fifteen diferent metabolites were annotated between the two groups. L-aspartic acid, ureidopropionic acid, adenosine monophosphate, L-tryptophan, and guanosine monophosphate were annotated at higher levels in human strains. Butyric acid, fumaric acid, isoleucine, leucine, and hippuric acid have been found in both human and animal strains. Certain metabolites were uniquely expressed in animal strains, with fold changes greater than 2. For example, putrescine modulates bioflm formation. Overall, this study provides biological insights into the substantial possible bacterial response refected in its macromolecular production, either at the proteomic or metabolomic level.