(2017) demonstrated that the co-culture of _Bacillus subtilis_ ZF3-1 with an indigenous bacterial consortium predominated by the _Burkholderiales_ order displayed an enhanced ability for crude oil degradation in aerobic conditions, which is likely to occur through the removal of \(n\)-alkane [17]. These studies indicate that mixtures of strains and/or contaminants enhance biodegradation. Mixtures of substrates affect biodegradation readability, resulting in degradation stimulation or inhibition of a single substrate in the mixture. Elucidating these substrate interactions is essential, as most polluted sites include a broad spectrum of compounds. Mutual inhibitory and enhancement effects induced by substrate mixtures in aerobic conditions have been demonstrated in the literature [18; 19; 20; 21; 22; 23; 24]. The biodegradation of halogenated organic compounds includes a variety of enzymatic mechanisms [25]. An essential route of halogenated organic degradation is dehalogenation, i.e., halogen-carbon bond cleavage, which can proceed either enzymatically or by spontaneous chemical dehalogenation of unstable intermediates [26]. Enzymatic dehalogenation involves multiple mechanisms (hydrolysis, reduction, or oxygen-dependent mechanisms) by which halogen atoms are removed. Hydrolysis includes halogen replacement through nucleophilic substitution by water derived from a hydroxyl group; reduction involves halogen replacement by hydrogen; and oxygenolytic dehalogenation, which is catalyzed by monooxygenase or dioxygenase, results in the incorporation of one or two atoms of molecular oxygen into the substrate [2; 26; 27; 28]. In the past decade, a multi-elemental isotope approach has been developed to understand complex degradation pathways, both biotic and abiotic. Dual compound specific isotope analysis (CSIA) may help us to understand degradation mechanisms, assist in differentiating between mechanisms, and clarify contaminant fate in the environment [29; 30; 31; 32; 33; 34]. Usually significant isotope fractionation occursduring bond cleavage and its magnitude depends on the degradation mechanism [32; 35]. However, isotopic analysis of bromine, a less commonly used technique in CSIA, has not been extensively investigated [29]. An application of a \({}^{13}\)C/\({}^{12}\)C-\({}^{81}\)Br/\({}^{79}\)Br isotope analysis to study the biotic transformation pathway of tribromoneopentyl alcohol by groundwater enrichment culture was demonstrated previously [36]. The enriched groundwater consortium was microbially characterized, but the strains involved in the process itself remained unidentified. The objectives the current work are to (1) isolate and culture the specific strains that are essential for the degradation of dibromoneopentyl glycol (DBNPG) and tribromoneopentyl alcohol (TBNPA), (2) examine the isolates' preference in degrading one compound over the other, and (3) use the isotopic tools (carbon and bromine fractionation) to elucidate whether the isolates are characteristic for the microbial activity in the original enrichment consortium. To this end, pure strains were isolated from a previously reported consortium [28], incubated in different combinations of TBNPA and DBNPG amendments, along with the use of auxiliary amendments (glucose, yeast extract, and vitamin mix) to determine the influence of supplements on degradation activity. Additionally, the \({}^{13}\)C/\({}^{12}\)C-\({}^{81}\)Br/\({}^{79}\)Br isotope analysis was carried out to compare the degradation mechanism of both compounds and compare the degradation mechanism by the co-culture of the isolated strains to the original consortium.