Semen is a complex fluid that contains spermatozoa and also functions as a dynamic bacterial microecosystem, comprising probiotics, pathogenic bacteria, and natural antimicrobial substances. Probiotic bacteria, such as Lactobacillus and Bifidobacterium, along with pathogenic bacteria like Pseudomonas aeruginosa and Escherichia coli, play significant roles in semen preservation and reproductive health. Studies have explored the impact of pathogenic bacteria on sperm quality, providing insights into the bacterial populations in mammalian semen and their influence on sperm function. These reviews highlight the delicate balance between beneficial and harmful bacteria, alongside the role of natural antimicrobial substances that help maintain this equilibrium. Moreover, we discuss the presence and roles of antimicrobial substances in semen, such as lysozyme, secretory leukocyte peptidase inhibitors, lactoferrin, and antimicrobial peptides, as well as emerging antibacterial substances like amyloid proteins. Understanding the interactions among probiotics, pathogens, and antimicrobial agents is crucial for elucidating semen preservation and fertility mechanisms. Additionally, the potential for adding probiotic bacteria with recombinant antibacterial properties presents a promising avenue for the development of new semen extenders. This review offers updated insights to understand the equilibrium of the bacterial microecosystem in semen and points toward innovative approaches for improving semen preservation.