The objective of this study was to determine the effect of metal ions on α-glucosidase, β-glucosidase, acid phosphatase, and phytase activity of L. reuteri. In the control group, L. reuteri MF14-C, MM2-3, SD2112, and DSM20016 produced the highest α-glucosidase (40.06 ± 2.80 Glu U/mL), β-glucosidase (17.82 ± 1.45 Glu U/mL), acid phosphatase (20.55 ± 0.74 Ph U/mL), and phytase (0.90 ± 0.05 Ph U/mL) respectively. The addition of Mg2+ and Mn2+ led to an increase in α-glucosidase activity of L. reuteri MM2-3 by 23.46 and 20.77 Glu U/mL respectively. The β-glucosidase activity of MM7 and SD2112 increased in the presence of Ca2+ by 9.65 and 9.85 and Fe2+ by 11.4 and 9.62 Glu U/mL respectively. Acid phosphatase produced by L. reuteri CF2-7F and MM2-3 increased in the presence of Mg2+, Ca2+, and Mn2+ by 13.53, 6.17, and 10.01 and 5.54, 5.92, and 3.98 Ph U/mL respectively. Phytase produced by L. reuteri MM2-3 increased in the presence of Mg2+ and Mn2+ by 0.26 and 0.38 Ph U/mL respectively. On average, Mg2+ and Mn2+ followed by Ca2+ led to the highest enhancement of tested enzymes. The effect of metal ions on the enzymatic activity of L. reuteri was found to be strain dependent. Thus, the enzymatic activity of L. reuteri could be enhanced by the addition of metal ions, with the specific selection of metal ions being necessary to maximize the level of the target enzyme.