Bromoxynil is an increasingly applied nitrile herbicide. Under aerobic conditions, hydration, nitrilation, or hydroxylation of the nitrile group commonly occurs, whereas under anaerobic conditions reductive dehalogenation is common. This work studied the isotope effects associated with these processes by soil cultures. The aerobic soil enrichment culture presented a significant increase in Stenotrophomonas, Pseudomonas, Chryseobacterium, Achromobacter, Azospirillum, and Arcticibacter, and degradation products indicated that nitrile hydratase was the dominant degradation route. The anaerobic culture was dominated by Proteobacteria and Firmicutes phyla with a significant increase in Dethiosulfatibacter, and degradation products indicated reductive debromination as a major degradation route. Distinct dual-isotope trends (δ13C, δ15N) were determined for the two routes: a strong inverse nitrogen isotope effect (εN = 10.56 ± 0.36‰) and an insignificant carbon isotope effect (εC = 0.37 ± 0.36‰) for the aerobic process versus a negligible effect for both elements in the anaerobic process. These trends differ from formerly reported trends for the photodegradation of bromoxynil and enable one to distinguish between the processes in the field.