The aim of this work was to demonstrate the importance of bacteriological investigation of milk from cows suffering from mastitis before the commencement of antibiotic treatment. Individual milk samples (10% of total milked cows; n = 36) from a dairy farm in Eastern Slovakia were tested for the presence of contagious and environmental mastitis pathogens. The following bacteria were identified in ten cows (27.77%): Streptococcus agalactiae, Staphylococcus lentus, Staphylococcus xylosus, Staphylococcus sciuri and Staphylococcus equorum. Bacteria such as Staphylococcus epidermidis, Pseudomonas aeruginosa, Bacillus spp. and members of the Enterobacteriaceae family were sporadically isolated and identified in other individual milk samples (n = 26). No Staphylococcus aureus or Streptococcus agalactiae were isolated in the milk from these cows. All isolates of Streptococcus agalactiae were resistant to amoxicillin- clavulanate, azithromycin, penicillin and oxacillin and partially sensitive to cephalothin and cefazolin antibiotics.