Biomass of Lactobacillus rhamnosus B 442 was subjected to the continuous electroporation using an electroporator with a flow chamber (length of 10 cm, distance between electrodes 0.25 cm, stream width 0.25 cm, flow speed 10 mL/min) to improve accumulation of calcium, magnesium and zinc in the cells. For all tested ions, the following parameters were applied: voltage of 250 V (E = 1 kV/cm), 570 V (E = 2.28 kV/cm), 950 V (E = 3.8 kV/cm), and 1400 V (E = 5.6 kV/cm, the positive control), a frequency of 10 Hz, a pulse width of 100 µs and 30 electrical pulses. The use of PEF increased the accumulation of magnesium, zinc and calcium by 39, 73 and 162%, respectively, compared to the control. Positive correlation was found between ion accumulation and membrane permeability for zinc and magnesium. For calcium, the initial increase in permeability resulted in higher ion accumulation, but with a further increase of this parameter at 3.8 kV/cm, its decrease was observed caused by a drop in cell viability. Total number of bacteria ranged from 1.67 × 108 (for the cultures supplemented with calcium) to 1.34 × 1012 cfu/mL (for the cultures supplemented with magnesium).