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One hundred and seventy-seven (177) bulk tank milk samples were analyzed with a commercially available real-time polymerase chain reaction kit and 11 (6.21%), 41 (23.16%), and 58 (32.77%) tested positive for <i>Mycoplasma bovis</i>, <i>Staphylococcus aureus,</i> and <i>Streptococcus agalactiae</i>, respectively. Statistical analysis revealed a significant relationship between the presence of <i>S. aureus</i> and <i>S. agalactiae</i>. Enumeration of somatic cells was performed in the same samples by flow cytometry. The somatic cell counts were found higher in <i>S. aureus</i> and <i>S. agalactiae</i> positive samples. No association was found between <i>M. bovis</i> presence and somatic cells counts. Low internal assay control Ct values were found to be related with high somatic cell counts. Noticeably, this is the first report for the presence of <i>M. bovis</i> in Cyprus. Therefore, its presence was confirmed by bulk tank milk culture, conventional PCR, and next generation sequencing. Furthermore, <i>M. bovis</i> was typed with multilocus sequencing typing and was allocated to sequence type 29 (ST 29). Real-time PCR in bulk tank milk samples is a useful tool to detect mammary infections, especially for neglected pathogens such as <i>M. bovis</i>.