To design cost-effective prevention strategies against mastitis in dairy cow farms, knowledge about infection pathways of causative pathogens is necessary. Therefore, we investigated the reservoirs of bacterial strains causing intramammary infections in one dairy cow herd. Quarter foremilk samples (n= 8056) and milking- and housing-related samples (n= 251; from drinking troughs, bedding material, walking areas, cow brushes, fly traps, milking liners, and milker gloves), were collected and examined using culture-based methods. Species were identified with MALDI-TOF MS, and selectedStaphylococcusandStreptococcusspp. typed with randomly amplified polymorphic DNA-PCR. Staphylococci were isolated from all and streptococci from most investigated locations. However, only forStaphylococcus aureus,matching strain types (n= 2) were isolated from milk and milking-related samples (milking liners and milker gloves).Staphylococcus epidermidisandStaphylococcus haemolyticusshowed a large genetic diversity without any matches of strain types from milk and other samples.Streptococcus uberiswas the onlyStreptococcusspp. isolated from milk and milking- or housing-related samples. However, no matching strains were found. This study underlines the importance of measures preventing the spread ofStaphylococcus aureusbetween quarters during milking.