Zero-valent iron (ZVI) application in groundwater remediation is limited by its vulnerability to passivation, which significantly decreases its surface reactivity. Both biological and chemical processes can potentially passivate ZVI, although the understanding of biological passivation is limited. This study was conducted in bench-scale reactors packed with fresh ZVI or ZVI pre-exposed to nitrate (NO3−) and in the presence or absence of a denitrifying bacterial enrichment (DNBE). The first-order rate coefficients (k) for NO3− reduction by ZVI in the presence and absence of DNBE were 0.20 and 0.09 s−1, respectively, suggesting that both ZVI and microbes contribute to NO3− removal. Abiotic reduction of nitrate was observed in reactors with trichloroethene (TCE) if ZVI was present; however, it resulted in reduced rates of TCE reduction (k = 0.29 s−1) when compared to reactors with fresh ZVI and no nitrate (k = 0.55 s−1). The TCE reduction efficiency decreased by 49% (k = 0.15 s−1) in the presence of DNBE, suggesting that microbial growth on ZVI or catalyzed oxidation of ZVI surface can inhibit TCE reduction by ZVI. Contrary to the presumption that denitrification may decrease ZVI passivation by nitrate, results from this study suggest that denitrifying bacteria actually exacerbate ZVI passivation.