A series of short-term experiments were carried out to assess the effect of phosphorus (P) and nitrogen (ammonium [NH4+-N] or nitrate [NO3−-N]) fertilization on P uptake and phosphatase activity in ryegrass and tall fescue cultivated under greenhouse conditions. Ryegrass or tall fescue plants were grown in an acidic Andisol in the presence or absence of P and increasing doses of NO3−-N or NH4+-N fertilizers. At the end of the experiment, soil phosphatase activity (P-aseRhiz), pH, and Olsen-P were determined in the rhizosphere soil. Plant biomass, P uptake, and root surface phosphatase (P-aseRoot) were also assayed for both plant species. Furthermore, soil incubation experiments at increasing doses of P, NO3−-N, or NH4+-N were performed to evaluate the fertilizer effect on soil phosphatase activity (P-aseBulk) and microbial biomass carbon in the bulk soil. In the absence of plants, P-aseBulk was inhibited and microbial biomass carbon was raised at increasing P supply levels. In the greenhouse experiments, P uptake by tall fescue was about 67% higher than that of ryegrass at low soil P availability, which suggests that tall fescue was less sensitive to P deficiency than ryegrass. For both plant species, P-aseRhiz did not vary as a consequence of P addition. On the other hand, fertilization with the highest NH4+-N dose strongly decreased soil pH and shoot P content, as well as it increased P-aseRoot activity. This fact denotes that P-aseRoot behaved as a strategic response parameter to P stress with insufficient impact on plant P nutrition in both plant species.