The Biological Safety Cabinet (BSC) is the most commonly used piece of engineering control in laboratories working with infectious agents or recombinant DNA, offering personnel, product, and environmental protection. BSCs are routinely decontaminated with formaldehyde, an effective biocide (which is then neutralized with ammonium bicarbonate). During these procedures, laboratory personnel vacate the room, and the decontamination technicians wear respiratory protection. Nevertheless, because formaldehyde is an irritant, a sensitizer, and a suspect human carcinogen, it is important to determine the potential laboratory levels. Using an electrochemical direct-reading instrument, this study examined ambient formaldehyde levels in five research laboratories undergoing decontamination. In four of the processes, formaldehyde levels were below the limit of detection (0.4 ppm) the entire time. However, the fifth one had two concentration peaks, one at 1.5 ppm, when formaldehyde was released into the cabinet, the other at 37.8 ppm, when the cabinet was opened up after the procedure. During the first peak, the ambient levels remained above the OSHA PEL of 0.75 ppm for 10 minutes. During the second spike, the levels stayed above the STEL of 2.0 ppm for 90 minutes and did not fall below the PEL for another 170 minutes. The first peak probably resulted from the cabinet not being tightly sealed. The second peak may have been the result of incomplete neutralization and the size of the cabinet relative to the room. These results indicate that while the usual controls are effective, some additional controls, such as measuring out the amount of ammonium bicarbonate more carefully (the current procedure involves making “eyeball” estimates), may still be needed to counteract and/or prevent such occasions. Further investigation is needed to determine the frequency of elevated formaldehyde levels during BSC decontamination procedures and the role that the ratio of cabinet-to-room size plays in determining these levels.