Lactococcus garvieae is a catalase-negative, Gram-positive coccus that occurs in pairs and short chains. It is present throughout the world and a causative agent of bovine mastitis. However, prior to writing, no case of mastitis caused by this bacterium had been reported in Poland. The aim of this research was to investigate the presence of L. garvieae in milk samples from Polish cows with inflammation of the mammary gland. Material and Methods: A total of 118 milk samples from 96 Holstein-Friesian cows were spread on blood agar plates for microbial analyses. Then analysis with matrix-assisted laser desorption/ionisation–time-of-flight mass spectrometry, biochemical identification and antibiotic susceptibility evaluation in the VITEK 2 system and a PCR with primers compatible with 16S ribosomal RNA of L. garvieae were performed. Results: The presence of three strains of L. garvieae was confirmed in milk samples. Analysis of antibiotic susceptibility showed the susceptibility of all isolates to moxifloxacin, erythromycin, linezolid, teicoplanin, vancomycin, tetracycline, tigecycline, chloramphenicol and trimethoprim-sulfamethoxazole. Lactococcus garvieae had high resistance to benzylpenicillin, clindamycin and rifampicin. One of the isolated strains was additionally resistant to tetracycline and had different minimum inhibitory concentrations. Conclusion: To the best of our knowledge, L. garvieae had not been identified as an aetiological factor of mastitis in cows in Poland before, and the presented research is the first report.