Phosphorus (P) immobilization by inactivating agents in the sediment of eutrophic lakes to reduce immediately available P in lake water is often crucial for mitigating nuisance eutrophication symptoms, such as cyanobacterial blooms. Macrophytes and phytoplankton, however, can directly utilize P from the sediment for growth. Accordingly, a comprehensive analysis of the P bioavailability in lake sediment amended with two promising P-inactivation agents, namely Phoslock® and drinking water treatment residue (DWTR), was investigated in both short- and long-term studies (20 and 180 d). Phosphorus-availability was assessed using six chemical extraction methods and Hydrilla verticillata and Microcystis aeruginosa growth tests. The results showed that Phoslock® and DWTR significantly reduced mobile P (NH4Cl and Na2S2O4/NaHCO3 extractable P) in lake sediment, while P bioavailability that was assessed by different methods showed considerable deviations. Interestingly, appropriate bioavailable P chemical extraction methods were determined based on linear correlation analysis, and further comparison indicated that reduction of bioavailable P by DWTR (<55% for macrophyte available P) and Phoslock® (<17% for cyanobacteria available P) were clearly less than the mobile P immobilization (>75%) at recommended dosages, which was probably caused by the capability of macrophyte and cyanobacteria to utilize various fractions of P (except the residual P) in amended sediment under proper illumination. Therefore, DWTR and Phoslock® can effectively reduce P release from lake sediment, but the potential bioavailable P may pose uncertainties for eutrophication control in lakes that typically have regular sediment re-suspension. Overall, an evaluation of the bioavailable P pool in the lake ecosystem should be essential for successful lake geo-engineering.