In the present investigation solvent displacement or nanoprecipitation, and emulsion/solvent evaporation methods were utilized to optimize poly(D,L-lactide-co-glycolide) nanoparticles for the vehiculization of the 5-fluorouracil. Formulation components from both the aqueous and organic phases, as well as, operating conditions were varied. Particles were characterized in terms of particle size and morphology, electrical properties, rheology, drug loading, stability, and drug release. Furthermore, in vitro cytotoxicity on human colon cells and different colon carcinoma cells was evaluated. Four types of nanoparticles were selected for drug loading, revealing differences between variables. Low viscosity values and their Newtonian behavior could assure the suitability of the nanoformulation for the intravenous route of administration. The greatest drug entrapment efficiency and best stability was achieved when the chemotherapeutic agent was incorporated into the internal aqueous phase of particles prepared by double emulsion/solvent evaporation. However, a more sustained drug release at pH 7.4 was possible when 5-fluorouracil was added to the external aqueous phase. These were the nanoformulations reporting the greatest antiproliferative efficacy compared with the free drug. The nanocarrier can optimize the antitumor activity of 5-fluorouracil, thus being a potential nanotool against colon cancer.