Mastitis in cattle is a worldwide problem in dairy farming with a major impact on animal welfare, life span and milk production. Interestingly, quarters of the same cow can differ in their susceptibility to mastitis. This indicates that susceptibility is defined on the quarter level rather than the cow level. To investigate whether milk characteristics play a role in quarter susceptibility to mastitis, we collected quarter milk samples of morning and evening milk, analysed the milk composition, and determined in vitro growth of Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli), two major mastitis causing pathogens, in these fresh milk samples. We found that almost all measured milk components differed significantly between quarters and that they showed a diurnal pattern. Furthermore, we observed that quantitative growth of S. aureus and E. coli differs significantly between morning milk and evening milk. Mixed model analyses revealed that replication of S. aureus was negatively associated with fat concentrations, S. aureus specific IgG1 antibodies, and morning milk. Replication of E. coli was negatively associated with fat concentrations, and positively associated with morning milk. Next, we studied whether the milk composition also influenced in vivo growth of S. aureus. Our results revealed that the numbers of S. aureus reisolated from milk post experimental intramammary inoculation was positively associated with the milk fat percentage and negatively associated with the S. aureus specific IgG1 titer pre-inoculation. Although the exact mechanism leading to the negative association between the S. aureus specific IgG1 titer and decreased S. aureus replication, both in vitro and in vivo, could not be identified, these finding supports the ongoing effort to develop a prophylactic vaccine against bovine S. aureus-induced mastitis and indicates that stimulation of the humoral immune response in milk against this bacterium might contribute to protection. Driven by this observation, we explored immunization strategies to increase the presence of antibodies in the bovine mammary gland. First, we determined the impact of vaccine administration via different routes and showed that vaccination via the subcutaneous route results in higher levels of specific, neutralizing antibodies compared to vaccination via the intramuscular, intranasal, and intramammary route. Next, we showed that subcutaneous immunizations resulted in higher antibody titers when administered near the udder compared to administration in the neck. Furthermore, we found that an Alum-Saponin-Oil based adjuvant stimulates the induction of specific antibody titers, favouring both IgG1 and IgG2 responses, and neutralization titers in both milk and serum more efficiently compared to adjuvants comprising Alum-Saponin or Alum-Oil. Moreover, while prime immunizations with Alum-Saponin-Oil near the udder resulted in high titer increases, immunization in the neck required a prime-boost regimen to induce similar titers. This implies that, when subcutaneously administered near the udder, a one-shot vaccination strategy with Alum-Saponin-Oil may be sufficient to efficiently increase intramammary antibody responses. Increased levels of functional antibodies allow the investigation of the role of antibodies in bovine intramammary immunity during S. aureus infections.