Melioidosis, caused by the Gram-negative bacterium Burkholderia pseudomallei, is an infectious disease with high rates of morbidity and mortality, which primarily affects low- and middle-income countries in South and Southeast Asia and Australia. The clinical manifestations of this disease are nonspecific and, therefore, rapid laboratory diagnosis is especially critical as appropriate management requires specific antimicrobials. This article aims to provide an overview of the current diagnostic methodologies, emerging technologies, susceptibility testing, and future perspectives for laboratory diagnosis of melioidosis. By examining conventional culture methods, mass spectrometry, antimicrobial susceptibility testing, antigen detection, molecular diagnostics, and serological assays, this article highlights the current challenges in accurately and cost-effectively diagnosing melioidosis in diverse clinical and resource-limited settings. A detailed analysis of current and future diagnostic methodologies will offer valuable insights for clinicians, researchers, and public health professionals. This review aims to influence clinical and laboratory guidelines for diagnosing melioidosis and future research directions.