Uranium (U), as the uranyl ion (UO22＋), is a widely distributed contaminant at several Department of Energy (DOE) sites, former war zones, and across the globe. Although many U remediation efforts depend on U-bacterial interactions, little information regarding U-bacterial interactions resolved at the molecular level exist. In this study, experiments were performed aimed at understanding the effect of molecular UO2 2＋ speciation on bacterial bioaccumulation and toxicity using an environmental Pseudomonas sp. isolate. Results showed that the charge and stability of UO22＋ species largely controlled the extent of UO22＋ bioaccumulation and UO2 2＋ toxicity, respectively. Further experimentation, including a combination of in vivo, in vitro, and in silico studies, revealed a specific mechanism of UO22＋ toxicity, the first to be reported. This mechanism involves the binding of UO2 2＋ to pyrroloquinoline quinone (PQQ), a cofactor present in a number of bacterial dehydrogenase enzymes. Based on the specific binding mode of UO22＋ to PQQ, it is hypothesized that the present work has direct implications for UO22＋ inhibition of flavoproteins, potentially extending the application of the findings of this work to eukaryotic systems. Recent trends suggest that U-related activity will increase in the near future, and therefore understanding fundamental interactions between UO22＋ and living systems is both an environmental and human health imperative.