The effect of metal ions on the enzymatic activity of Lactobacillus reuteri was studied. The enzymatic activity was determined spectrophotometrically using the corresponding substrate. 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 enhance α-glucosidase produced by L. reuteri MM2-3 by 113.6% and 100.6% respectively. α-Glucosidase produced by MF14-C and CF2-7F was decrease in the presence of K+ by 65.8 and 69.4% respectively. β-Glucosidase activity of MM7 and SD2112 increased in the presence of Ca2+ (by 121.8 and 129.8%) and Fe2+ (by 143.9 and 126.7%) respectively. Acid phosphatase produced by L. reuteri CF2-7F and MM2-3 was enhanced in the presence of Mg2+, Ca2+ or Mn2+ by (94.7, 43.2, and 70.1%) and (63.1, 67.8, and 45.6%) respectively. On the other hand, Fe2+, K+, and Na+ caused only slight increase or decrease in acid phosphatase activity. Phytase produced by L. reuteri MM2-3 was increase in the presence of Mg2+ and Mn2+ by 51.0 and 74.5% respectively. Ca2+ enhanced phytase activity of MM2-3 and DSM20016 by 27.5 and 28.9% respectively. The addition of Na+ or Fe2+ decreased phytase activity of L. reuteri. On average, Mg2+ and Mn2+ followed by Ca2+ led to the highest enhancement of the tested enzymes. However, the effect of each metal ion on the enzymatic activity of L. reuteri was found to be a strain dependent. Therefore, a maximized level of a target enzyme could be achieved by selecting a combination of specific strain and specific metal ion.