A multi-tier approach for use in a mailroom for detecting bio-threats conveyed by mail. In a first tier procedure, mail entering the mailroom is continuously automatically screened at a mail screening station to detect a potential bio-threat contamination. If a potential bio-threat is detected, an alarm signal is generated, a sample of the potential bio-threat is collected, and a second tier procedure is initiated. The second tier procedure uses a manual test, such as a nucleic acid amplification and detection assay to detect any of a plurality of different specific bio-threats in the sample. If a specific bio-threat is found, appropriate steps are taken to limit spread of the bio-threat and exposure of personnel. A third tier procedure uses a manual assay to check for a specific toxin in background samples collected over a predefined time interval at each mail screening station and in the air handling system for the mailroom.