Containment of hazardous aerosols in biological safety cabinets occurs by air barriers, physical barriers, and high-efficiency particulate air (HEPA) filtration. Air barriers provide containment by providing directional airflow from the laboratory, past the researcher, and into the cabinet via the work opening. Hazardous aerosols generated during experimental procedures inside the cabinet become captured and carried by the flow of air, and trap within HEPA filters. Some BSCs provide protection for experimental procedures by providing uniform, unidirectional HEPA filtered air, referred to as laminar airflow, continuously flowing over the work area. Laminar airflow minimizes turbulence inside the cabinet, allowing for immediate removal of contaminants generated by the procedures. Disruption of the airflow patterns in the cabinet can compromise the integrity of the containment provided by air barriers, and objects within the cabinet can disrupt airflow patterns. Therefore, air barriers provide only partial containment, and you should not use them solely to contain highly toxic or infectious materials.

Physical barriers are impervious surfaces such as metal sides, glass panels, rubber gloves and gaskets, which physically separate the experimental procedures from the researcher. Biological safety cabinets incorporating physical barriers (e.g. Class III BSCs), and not relying on air barriers, can be used for higher risk agents since compromised containment is less likely.

HEPA filters have a filtration efficiency of 99.97% for thermally generated monodisperse dioctylphthalate (DOP) 0.3 µm diameter particles. Because of their high efficiency, HEPA filters in biological safety cabinets can remove virtually all particulates, including hazardous microbiological and chemical aerosols, in the air stream passing through the filter. All biological safety cabinets have exhaust filters that remove contaminants as air discharges from the cabinet. Some types (discussed below) also have supply HEPA filters to provide clean air to the work area. HEPA filters are not effective in capturing chemical vapors, and are not considered protective against gases or vapor-phase solids/liquids.