Engineering strategies to reduce greenhouse gases (GHGs) emissions by inhibiting methanogenesis in oil sands tailings have rarely been examined. In this study, we explored the potential impact of chemical treatment (lime) and biological treatment using enzymes (lysozyme and protease) on inhibiting methane emissions from tailings. Overall, treatment with protease 3%, lysozyme 3%, and lime 5000 ppm reduced CH4 production (by 52%, 28%, and 25%, respectively) and were weakly associated with the archaeal abundance. Enzymes treatment resulted in a higher reduction in CH4 production compared with lime treatment. A 3% lysozyme treatment suppressed CH4 production (the change in methane was 0.48 mmol) and reduced the degradation of hexane throughout the experiment. Similarly, 3% protease suppressed CH4 production throughout the experiment (the change in methane was 0.78 mmol), which could be attributed to the pH reduction to pH 4.9 at week 23 resulting from the formation of volatile fatty acids. Another possible mechanism could be the formation of toxic compounds, such as high nitrogen content, after protease treatment that inhibited the microbial community. The toxicity effect to Vibrio fischeri was greater with lysozyme 3% and protease 3% treatment than with lime treatment (124 TU and 76 TU, respectively). Lime treatment resulted in the highest reduction in 16S rRNA gene copies from 5.7 × 106 cells g−1 (control) to 2.7 × 105, 1.71 × 105, and 1.4 × 105 cells g−1 for 1600, 3500, and 5000 ppm treatments, respectively. This study supports further work to examine and determine the optimum conditions (e.g., enzyme and lime dosages) for CH4 inhibition.