Significant amounts of manure are produced in the USA; however, information on the changes in ecosystem services related to soil biogeochemical cycling for agroecosystems supported with organic amendments such as manure is limited. A multi-location field study was initiated in Colorado (CO), Kansas (KS) and Kentucky (KY), USA in loam soils to evaluate the effects of manure and tillage practices on enzyme activities that are key to biogeochemical cycling such as β-glucosidase (C cycling), α-galactosidase (C cycling), β-glucosaminidase (C and N cycling) and phosphomonoesterases (P cycling). The treatments were as follows: (i) two years of beef manure applications to a fine sandy loam at different rates (control: 0, low: 34 kg N ha⁻¹ and high: 96 kg N ha⁻¹) and tillage practices in CO; (ii) three years of beef manure applications to a silt loam at different rates (0, low: 67 kg N ha⁻¹ and high: 134 kg N ha⁻¹) and tillage practices in KS and; (iii) three years of poultry and dairy manure applications to a silt loam with different tillage practices at the same rate (403 kg N ha⁻¹) in KY. Tillage practices (none vs. conventional) had no effect on the enzyme activities. Principal Component Analyses (PCA) grouped all enzyme activities with the high beef manure application rate after the first year in CO at 0–5 cm. By the second year, the low and high beef manure rates differed in enzyme activities for the KS soil with no difference between the low rate and control in CO. Since the first year of the KY study, acid phosphatase activity was greater in the poultry treated soil compared to dairy or the control; whereas, C cycling enzyme activities were similar in soil treated with dairy or poultry manure. For all studies, PCAs for soil samples from 5–10 cm depth did not reveal treatment separation until the second year, i.e., only high application rate differed from the other treatments. Results of the study indicated significant responses in C and P cycling enzyme activities to manure applications within two years, suggesting potential benefits to soil biogeochemical cycling essential for the productivity of agroecosystems supported with organic fertilizers.