The gastrointestinal tract houses a reservoir of bacterial-derived enzymes which can directly catalyze the metabolism of drugs, dietary elements, and endogenous molecules. Both host and environmental factors may influence this enzymatic activity, with the potential to dictate the availability of the biologically-active form of endogenous molecules in the gut and influence inter-individual variation in drug metabolism. We aimed to investigate the influence of the microbiota, and the modulation of its composition, on fecal enzymatic activity. Intrinsic factors related to the host, including age, sex, and genetic background, were explored. Fecalase, a cell-free extract of feces, was prepared and used in a colorimetric-based assay to quantify enzymatic activity. To demonstrate the functional effects of fecal enzymatic activity, we examined ß-glucuronidase-mediated cleavage of serotonin ß-D-glucuronide (5-HT-GLU) and the resultant production of free 5-HT by HPLC. As expected, ß-glucuronidase and ß-glucosidase activity were absent in germ-free mice. Enzymatic activity was significantly influenced by mouse strain and animal species. Sex and age significantly altered metabolic activity with implications for free 5-HT. ß-glucuronidase and ß-glucosidase activity remained at reduced levels for nearly two weeks after cessation of antibiotic administration. This effect on fecalase corresponded to significantly lower 5-HT levels as compared to incubation with pre-antibiotic fecalase from the same mice. Dietary targeting of the microbiota using prebiotics did not alter ß-glucuronidase or ß-glucosidase activity. Our data demonstrate that multiple factors influence the activity of bacterial-derived enzymes which may have potential clinical implications for drug metabolism and the deconjugation of host-produced glucuronides in the gut.