This work aims at fabricating 5-fluorouracil (5-FU)-loaded poly (lactic-co-glycolic) acid nanoparticles (PLGA NPs) using a microfluidic (MF) technique, with potential for use in colorectal cancer therapy. In order to achieve 5-FU-loaded NPs with an average diameter of approximately 119 nm, the parameters of MF process with fork-shaped patterns were adjusted as follows: the ratio of polymer to drug solutions flow rates was equal to 10 and the solution concentrations of PLGA as carrier, 5-FU as anti-cancer drug and poly (vinyl alcohol) (PVA) as surfactant were 0.2 (% w/v), 0.01 (% w/v) and 0.15 (% w/v), respectively. In this way, a drug encapsulation efficiency of approximately 95% into the PLGA NPs was obtained, due to the formation of a hydrodynamic flow focusing phenomenon through the MF chip. A performance evaluation of the NP samples in terms of the drug release, cytotoxicity and cell death was carried out. Finally, by analyzing the results after induction of cell death and 4′, 6-diamidino-2-phenylin-dole (DAPI) staining, MF-fabricated NPs containing 5-FU [0.2 (% w/v) of PLGA] revealed the dead cell amounts of 10 and 1.5-fold higher than the control sample for Caco2 and SW-480, respectively.