Nitrogen is a macronutrient, and, in its ammonium form, it is easily assimilated by most microorganisms. For assessing the effect of NH4+ in bioleaching environments, we designed column tests in both suitable and stressed conditions—specifically, low temperatures and inhibitory sulfate concentrations. We observed differential effects between suitable and stressed environments. The addition of NH4+ generated a significant increase in the total bacterial number and a slight increase in the oxidation activity in a suitable environment. The cease in NH4+ amendment has a higher impact on the archaea compared to the bacterial population. This interruption in NH4+ feeding also impacted the ferrous iron oxidation activity. Based on the gene expression analysis, we realized that the nifH gene was overexpressed by Leptospirillum at NH4+ levels lower than 10 mg L−1. Moreover, in bioleaching columns at low temperatures, the total bacterial and archaeal cell numbers depended on the NH4+ levels. Higher NH4+ levels in those columns enhance the Most Probable Number (MPN) of Fe-oxidizing microorganisms and the oxidation activity; therefore, the lag phase in the Eh increase was shorter and the Cu recovery higher. The redox potential decreased in the pregnant leaching solution (PLS) of the column after the turn-off of NH4+ feeding. The ion concentration in 9 m columns supplemented with 40 mg L−1 of NH4+ decreased from 37 to 5 mg L−1 after 50 days of amendment cease. In column tests, at high sulfate concentrations, the NH4+ amendment significantly increases microbial activity and Cu recovery.