The human body is vastly colonised by microorganisms, whose impact on health is increasingly recognised. The human genital tract hosts a diverse microbiota, and an increasing number of studies on the male genital tract microbiota suggest that bacteria have a role in male infertility and pathological conditions, such as prostate cancer. Nevertheless, this research field remains understudied. The study of bacterial colonisation of the male genital tract is highly impacted by the invasive nature of sampling and the low abundance of the microbiota. Therefore, most studies relied on the analysis of semen microbiota to describe the colonisation of the male genital tract (MGT), which was thought to be sterile. The aim of this narrative review is to present the results of studies that used next-generation sequencing (NGS) to profile the bacterial colonisation patterns of different male genital tract anatomical compartments and critically highlight their findings and their weaknesses. Moreover, we identified potential research axes that may be crucial for our understanding of the male genital tract microbiota and its impact on male infertility and pathophysiology.