β-Proteobacteria is one of the most abundant phylum in soils, including autotrophic and heterotrophic ammonium-consumers with relevance in N circulation in soils. The effects of high-temperature events and phytosanitary treatments, such as copper amendments, on soil bacterial communities relevant to N-cycling remain to be studied. As an example, South Portugal soils are seasonally exposed to high-temperature periods, the temperature at the upper soil layers can reach over 40 °C. Here, we evaluated the dynamics of mesophilic and thermophilic bacteria from a temperate soil, in particular of heterotrophic β-Proteobacteria, regarding the ammonium equilibrium, as a function of temperature and copper treatment. Soil samples were collected from an olive orchard in southern Portugal. Selective enrichments were performed from samples under different conditions of temperature (30 and 50 °C) and copper supplementation (100 and 500 µM) in order to mime seasonal variations and phytosanitary treatments. Changes in the microbial communities under these conditions were examined by denaturing gradient gel electrophoresis, a molecular fingerprint technique. At moderate temperature--30 °C--either without or with copper addition, dominant members were identified as different strains belonging to genus Massilia, a genus of the Oxalobacteraceae (β-Proteobacteria), while at 50 °C, members of the Brevibacillus genus, phylum Firmicutes were also represented. Ammonium production during bacterial growth at moderate and high temperatures was not affected by copper addition. Results indicate that both copper and temperature selected specific tolerant bacterial strains with consequences for N-cycling in copper-treated orchards.