Co-application of biosolids and water treatment residuals (WTR) land has not been extensively studied but may be beneficial by sorbing excess biosolid-borne or soil phosphorus (P) onto WTR, reducing the likelihood of off-site movement. Reduction of excess soil P may affect the role of specific P-cleaving enzymes. The research objective was to understand the long-term effects of single co-applications and the short-term impacts of repeated co-applications on soil acid phosphomonoesterase, phosphodiesterase, pyrophosphatase, and phytase enzyme activities. Test plots were 7.5 x 15 m with treatments consisting of three different WTR rates with a single biosolids rate (5, 10, and 21 Mg WTR ha-1; 10 Mg biosolids ha-1) surface co-applied once in 1991 or reapplied in 2002. Control plots consisted of those that received no WTR-biosolids co-applications and plots that received only 10 Mg biosolids ha-1. Plots were sampled to a 5-cm depth in 2003 and 2004, and soil phosphatases and phytase enzyme activities were measured. Soil phosphodiesterase activity decreased in WTR-amended plots, and pyrophosphatase activity decreased with increasing WTR application rates. In contrast, acid phosphatase and phytase activity increased with WTR addition, with WTR application possibly triggering a deficiency response causing microorganisms or plants to secrete these enzymes. Biosolids and WTR co-applications may affect enzymatic strategies for P mineralization in this study site. Reductions in phosphodiesterase activity suggest less P mineralization from biomass sources, including nucleic acids and phospholipids. Increased acid phosphatase and phytase activities indicate that ester-P and inositol-P may be important plant-available P sources in soils amended with WTR.