Enzymatic hydrolysis of complex components of residual materials, such as food waste, is a rate-limiting step that conditionates the production rate of biofuels. Research into the anaerobic degradation of cellulose and starch, which are abundant components in organic waste, could contribute to optimize biofuels production processes. In this work, a lab-scale anaerobic semi-continuous hydrolytic reactor was operated for 171 days using food waste as feedstock; the effect of Ba2+ dosage over the activity of five hydrolytic enzymes was also evaluated. No significant effects were observed on the global performance of the hydrolytic process during the steady-state of the operation of the reactor, nevertheless, it was detected that Ba2+ promoted β-amylases activity by 76%, inhibited endoglucanases and α-amylases activity by 39 and 20%, respectively, and had no effect on β-glucosidases and glucoamylases activity. The mechanisms that rule the observed enzymatic activity changes remain unknown; however, the discussion in this paper provides hypothetical explanations for further research.