The use of hydrogen peroxide as a sanitant in isolators and other barrier systems is well documented. To confirm that the isolator maintains a germ-free environment between decontamination cycles, microbiological air monitoring is performed after the sanitation and aeration cycles. In this study, we have shown that residual levels of hydrogen peroxide as low as 1 ppm can remain in the isolator and inhibit the growth of microorganisms after concentration on agar media. This lingering hydrogen peroxide can make accurate microbiological air monitoring difficult and can even cause false negative test results. To solve this issue, we have developed a new media that can mediate the effects of residual peroxide and prevent false negative test results. Initially, catalase was tested as a neutralizing agent but proved not to be efficient enough. Instead, 1% pyruvate was added, which was able to tolerate as much as 15 ppm Vaporous Hydrogen Peroxide (VHP) and ensured growth promotion of Staphylococcus aureus, Pseudomonas aeruginosa, Micrococcus luteus, Bacillus subtilis, Candida albicans and Aspergillus niger. The 1% pyruvate retained its neutralizing activity for Micrococcus luteus at up to 100 ppm VHP. Raising the pyruvate concentration to 5% pyruvate enabled neutralization of up to 300 ppm VHP, permitting subsequent growth of Micrococcus luteus on agar media.