Brucellosis is considered one of the most serious zoonotic diseases worldwide. This disease affects both human and animal health, in addition to being one of the most widespread zoonotic illnesses in the Middle East and Northern Africa. Human brucellosis generally presents in a diverse and non-specific manner, making laboratory confirmation of the diagnosis critical to the patient's recovery. A coordinated strategy for diagnosing and controlling brucellosis throughout the Middle East is required, as this disease cannot be known to occur without reliable microbiological, molecular, and epidemiological evidence. Consequently, the current review focuses on the current and emerging microbiological diagnostic tools for the early detection and control of human brucellosis. Laboratory assays such as culturing, serology, and molecular analysis can frequently be used to diagnose brucellosis. Although serological markers and nucleic acid amplification techniques are extremely sensitive, and extensive experience has been gained with these techniques in the laboratory diagnosis of brucellosis, a culture is still considered to be the "gold standard" due to the importance of this aspect of public health and clinical care. In endemic regions, however, serological tests remain the primary method of diagnosis due to their low cost, user-friendliness, and strong ability to provide a negative prediction, so they are commonly used. A nucleic acid amplification assay, which is highly sensitive, specific, and safe, is capable of enabling rapid disease diagnosis. Patients who have reportedly fully healed may continue to have positive molecular test results for a long time. Therefore, cultures and serological methods will continue to be the main tools for diagnosing and following up on human brucellosis for as long as no commercial tests or studies demonstrate adequate interlaboratory reproducibility. As there is no approved vaccine that prevents human brucellosis, vaccination-based control of animal brucellosis has become an important part of the management of human brucellosis. Over the past few decades, several studies have been conducted to develop vaccines, but the problem of controlling brucellosis in both humans and animals remains challenging. Therefore, this review also aims to present an updated overview of the different types of brucellosis vaccines that are currently available.