Biodegradable polymeric nanoparticles (NP) are promising delivery systems for photosensitizers (PS). A hydrophobic PS, the meso-tetra(p-hydroxyphenyl)porphyrin (m-THPP) was encapsulated into NP made of poly(D,L-lactide-co-glycolide) using an emulsification-diffusion method, and poly(vinyl alcohol) (PVAL) as stabilizing agent during the emulsification step. Three batches of NP with mean diameters of 117, 285, and 593 nm, respectively, were prepared. NP of 117 nm exhibited the highest rate of reactive oxygen species production and the fastest m-THPP release determined by the transfer of m-THPP to serum proteins in vitro. Similarly, the 117 nm batch exhibited the highest in vivo photodynamic activity, as established in the chick embryo chorioallantoic membrane model. Our data suggest that m-THPP release before illumination is an important step for in vivo photodynamic activity especially for large NP. The NP size and the amount of residual PVAL at the NP surface were shown to control both the PS release and activity.