The separate and combined effects of moderate levels of acid load and Cu-Ni deposition on humus (F/H layer) microbial community structure were examined six growing seasons after the start of an artificial irrigation experiment. A 22-factorial design with acid load (pH 3.1) and Cu-Ni addition was used. The acid application resulted in acidification as defined by humus chemistry, including a decrease in the concentration of base cations and an increase in the concentration of H+, Al and Fe, leading to a decrease in humus pH from 4.10 to 3.95. The metal additions resulted in a 2–3-fold increase in humus total Cu and Ni concentrations. The treatments did not affect the total microbial biomass, measured by substrate-induced respiration, or the basal respiration rate. However, the microbial community structure determined by phospholipid fatty acid (PLFA) analysis showed that the acid load affected the bacterial part of the community. The relative amount of branched PLFAs common to Gram-positive bacteria increased with decreasing humus pH, and the bacterial community was also adapted to more acidic conditions. The metal addition alone did not cause clear changes in the microbial community structure or bacterial tolerance to Cu or Ni. The combined acid and metal treatment induced similar changes in the microbial community structure as the acid treatment alone.