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The objective of this study was to evaluate the effectiveness of different antibiotics against mastitis causing microorganisms in lactating dairy cows in and around Nitra region, Slovakia. Milk samples from quarters were cultured and bacteriologically evaluated. All the bacteria isolated through microbiological procedures were subjected to antimicrobial susceptibility test by disc diffusion method to a large number of antibiotics. The results revealed higher sensitivity against tetradelta (100 % of Streptococcus agalactiae and uberis, Escherichia coli (E. coli), Coagulase Negative Staphylococci (CNS)), (97.37 % of Staphylococcus aureus) with highest number of bacterial isolates, followed by enrofloxacin (100 % of Strep. agalactiae and uberis), (97.37 % Staph. aureus), (97.14 % of (CNS), cefalexin + kanamycin (100 % of Strep. agalactiae and uberis), (97.14 % of CNS), (96.0 % of E. coli) and amoxicillin + clavulanat (100 % of Strep. agalactiae and uberis), (98.57 % of CNS), (94.74 % of Staph. aureus), (94.0 % of E. coli). Maximum resistance was observed against penicillin (96.0 % of E. coli) and streptomycin (66.67 % of Strep. uberis). In conclusion, in vitro antibiogram studies of bacterial isolates revealed higher sensitivity for tetradelta, enrofloxacin, a combination of cefalexin plus kanamycin and amoxicillin plus clavulanat acid.