Semen contamination is one of the main issues of concern in livestock industry. While the presence of some bacteria in semen is considered as normal, high bacterial loads detrimentally affect sperm quality and fertilizing ability, thus having a negative repercussion on the efficiency of artificial insemination. For this reason, the present chapter focuses on the methods used for the assessment of semen contamination in farm animals. Routinely, bacterial culture is performed to assess the overall degree of contamination in sperm samples; in some cases, however, the identification of bacterial species is needed to determine the optimal antibiotic composition of semen preservation media and address whether those bacteria have developed antibiotic resistance. In addition, other techniques, such as scanning electron microscopy, can be used to evaluate sperm-bacteria interaction. This chapter also discusses the strategies to prevent bacteria contamination in semen. As the ejaculate is not a sterile fluid, media for preservation usually include, as aforementioned, antibiotics to avoid bacterial growth. Because of the mounting restriction in the usage of antibiotics, which is due to the increase of bacterial resistance, other approaches to prevent bacterial contamination of semen include preservation at low temperatures (5 °C) and the inclusion of alternative molecules such as antimicrobial peptides.