The objective of this work was to study the enzymatic activity of Lactobacillus in a sweet potato-based medium (SPM). SPM was formed using an extract from baked sweet potatoes and supplemented with 4 g/L of each nitrogen source (beef extract, yeast extract, and proteose peptone #3). Lactobacillus strains were grown in SPM and MRS for 16 h at 37 °C and then plated to determine the final bacterial populations. The strains were screened spectrophotometrically for α-glucosidase, β-glucosidase, acid phosphatase, and phytase using the corresponding substrate. Our results showed no significant (p > 0.05) differences in the final bacterial populations of Lactobacillus strains grown in SPM and MRS. All Lactobacillus strains, except L. reuteri, showed similar α-glucosidase and β-glucosidase activity in SPM and MRS. L. reuteri showed lower α-glucosidase and higher β-glucosidase activity in SPM compared to MRS. Acid phosphatase activity of Lactobacillus in SPM was similar to that in MRS except for L. reuteri SD2112 having higher acid phosphatase in SPM than MRS. In regard to phytase, all strains showed higher activity in SPM than MRS. Strains of L. reuteri showed the highest enzymatic activity of α-glucosidase, acid phosphatase, and phytase whereas L. delbrueckii subsp. bulgaricus SR35 showed the highest β-glucosidase activity. Thus, the growth of Lactobacillus in a SPM could result in enhanced or comparable level of enzymatic activity while showing similar growth compared to MRS.