Marginal regions are relatively large part of the area Slovakia which in terms of the economy breeding ruminants can efficiently produce animal commodities only occasionally. Geographic, social and economic stability of these regions is strongly influenced by breeding of ruminants with market milk production [1].  Mastitis is a disease complex that assumes highest clinical and economic significance in milk animals particularly medium to high yielding dairy cattle, usually in and around periparturient period [2]. The objective of this study was to evaluate the effectiveness of different antibiotics against mastitis causing microorganisms during first month of lactation in two herds of 230 and 310 dairy cows situated in marginal parts of Slovakia. Milk samples from quarters were cultured and identified bacteria were subjected to antimicrobial susceptibility test by disc diffusion method to a large number of antibiotics. The prevalence of mastitis in the monitored herds of dairy cows was 26.1% to 17.6%, respectively. A total of 1663 milk samples from udder quarters were investigated, 446 (21.3%) samples were positive. No pathogens were isolated from 1663 (78.4%) milk samples. From all tested bacteria Staphylococcus spp. and Streptococcus spp. which were isolated from subclinical and clinical mastitis, were found amoxicillin + clavulanat and tetradelta to be most effective drug followed by ceftiofur and rifaximin. The significant difference was confirmed between the Staph. aureus and coagulase-negative staphylococci (CoNS) isolates with respect  to their susceptibility to the various  antibiotics. Antibiotic susceptibility tests should be done to determine the effectiveness of drug that can be used for successful treatment of diseases. Proper isolation and identification of the causative organism play significant role in prevention and control of the diseases.