Biosafety Level 2 (BSL-2) facilities are specialized laboratories that ensure the safe handling of medium-risk biological agents, mainly risk group (RG) 2 organisms that pose a risk to personnel and the environment. These laboratories are essential to advance research, diagnostics and biotechnological applications involving pathogens associated with human disease. Key features of BSL-2 facilities include restricted access for authorized personnel, mandatory use of personal protective equipment (PPE) such as lab coats, gloves and face shields, and containment measures such as biosafety cabinets (Class II) to minimize exposure to infectious agents. Laboratory ventilation systems are designed to direct airflow from clean to contaminated areas to prevent the release of pathogens to the outside environment. Safety equipment, including autoclaves, eyewash stations and safety showers, support rapid response to potential incidents (Biosafety Manuals, ICMR Publications, 2024). These laboratories play a central role in microbiology, virology, diagnostic testing, teaching and provide essential infrastructure for advancing scientific and public health goals. Compared to other biosafety levels, BSL-2 laboratories strike a balance between operational efficiency and rigorous biosafety protocols. The robust security practices combined with a controlled environment make BSL-2 facilities an indispensable part of modern biological research. They protect laboratory personnel, the environment and public health while enabling breakthrough discoveries in the life sciences. The Department of Biotechnology, India has set specific guidelines for BSL-2 laboratories, which include design specifications, required safety equipment, operational practices and safety protocols (Guidelines, DBT BSL2 and BSL3 and Certification of BSL3 facility 2020). In addition, these laboratories must adhere to international safety standards set by organizations such as the World Health Organization (WHO) and the Centers for Disease Control and Prevention (CDC). The guidelines ensure that facilities can safely work with genetically modified organisms, handle biological material and conduct research that could use RG 2 pathogens. The most important points of the BSL2 guidelines are summarized in this chapter.