Amendment with aluminum sulfate (alum) is considered a best management practice for benefits in poultry production and increased nutrient retention in the litter. However, little is known about how long-term applications of alum-treated litter will affect soil organisms and C. Soil from grass plots amended annually for 8 and 9 years with alum-treated or untreated poultry litter applied at 2.24 (low rate) and 8.96 (high rate) Mg litter ha−1, ammonium nitrate (65 or 260 kg N ha−1), and an unamended control were sampled before, 10 days, 1 month, and 6 months after applications. There was no fertilizer × rate × sampling time interaction for dissolved organic C (DOC), total soil C, microbial biomass C, or dehydrogenase activity. Total C was 22 to 23 mg C g−1 in soil receiving the high rate of alum-treated and untreated poultry litter compared with 15 to 18 mg C g−1 in other treatments, but DOC was higher at 60.2 μg C g−1 in soil receiving the high rate of alum-treated litter compared with DOC at 47.9 μg C g−1 in soil receiving untreated poultry litter. The high rate of alum-treated litter increased microbial biomass C (338.2 μg C g−1) compared with the low rate of alum-treated litter, the control, and the high rate of ammonium nitrate, whereas the high rate of ammonium nitrate (188.5 μg C g−1) decreased microbial biomass C compared with the high rate of either poultry litter or the low rates of untreated litter and ammonium nitrate. The high rate of ammonium nitrate also decreased dehydrogenase activities and pH compared with all other treatments but had the highest DOC at 82.0 μg C g−1. The high rate of both poultry litters increased soil C, but DOC concentrations, microbial biomass, and pH indicate that the soil microbial community differs after 8 and 9 years of alum-treated compared with untreated poultry litter applications.