The microbial role in mobilization of trace elements from land-applied wastewater sludge is not well-defined. Our study examined the leachability of trace elements (Cd, Cr, Cu, Mo, Ni, P, Pb, S, and Zn) from dewatered sludge as affected by treatments designed to alter microbial activity. Different levels of microbial activity were achieved by incubating sludge columns at 4, 16, 28, and 37 °C and by the addition of AgNO3 biocide at each temperature. Columns (with inert glass bead support beds) were subjected to six consecutive incubation−leaching cycles, each consisting of 7.3-d incubation followed by 16-h leaching with synthetic acid rain. Glucose mineralization tests were used to assess overall microbial activity. Significant acidification and trace element leaching occurred when conditions favored microbial activity (16 and 28 °C). Extent of mobilization was element-specific with Zn, Ni, and Cu showing the greatest mobilization (99, 67, and 57%, respectively). Mobilization was reduced but still substantial at 4 °C. Conditions that best inhibited microbial activity (37 °C or biocide at any temperature) resulted in the least mobilization. Characterization of enrichments performed using thiosulfate as the sole energy source revealed the presence of both known and putative S-oxidizing bacteria in the sludge. The results suggest that microbial acidification via S oxidation can mobilize trace elements from sludge. Elemental mobility in field situations would also be governed by other factors, including the capacity of soil to buffer acidification and to adsorb mobilized elements.