Aims: We employed a combination of diagnostic tests including single cervical tuberculin test (SCT), rapid lateral-flow test (RT), TB-Feron, conventional PCR and culture to investigate the prevalence of Mycobacterium bovis infections in dairy cattle under the intensive dairy production system in Egypt. Methods and Results: In total, 2710 dairy cows from 11 herds in six Governorates were tested by SCT and 444 (16
4%) were TB reactors. Only 65 cows responded to M. bovis antigen by RT and TB-Feron. A postmortem examination showed that 49 (75
4%) of slaughtered cows have visible lesions. Testing of 215 out 444 SCT reactor cows by culture and PCR using blood and milk samples revealed that M. bovis were more frequently identified in the blood (6
1–20
5%) than milk (2
3–5
6%) samples. Additionally, in this study, we investigated the prevalence and potential risk factors associated with M. bovis infection in dairy farm workers. Overall, 100 dairy farm workers were tested using QuantiFERON-TB Gold In-Tube test to detect Mycobacterium tuberculosis complex (MTC) and 35 (35%) were positive. In all, 23 (23%) of MTC positive were M. bovis positive using PCR. Mycobacterium bovis positive cases were associated with workers who had respiratory signs and did not wash or disinfect their hands after handling cows. Conclusions: The prevalence of M. bovis in dairy cows and dairy farm workers under the intensive dairy production system in Egypt is high. It is therefore essential to disseminate effective prevention and control measures to prevent the spread of M. bovis between dairy cows and dairy workers. Significance and Impact of the Study: This study revealed that the use of RT or TB-Feron as an ancillary test of SCT reactor cows resulted in a significant reduction in the SCT false-positive slaughtered cows. A high prevalence of M. bovis infection among farm workers provides evidence of occupational risk in the intensive dairy production system in Egypt.