The nanoencapsulation of chlorogenic acid (CGA) by nanospray drying was investigated as a protective bioactive delivery strategy. This study aimed to develop CGA-loaded nanoparticles (NPs) for potential application in colorectal cancer (CRC) therapy using the SW480 and HT-29 cell lines. Eight formulations were prepared with biopolymers—maltodextrin (MD), arabic gum (AG), starch (S), carboxymethyl cellulose (CMC), and hydroxypropyl methylcellulose (HPMC)—and the surfactant polysorbate 80 (PS80). The formulations were transformed into fine powders through nanospray drying and comprehensively characterized using physicochemical techniques. In vitro cytotoxicity and proliferation assays were performed using the MTT method across CGA concentrations from 0 to 400 μM. In addition, cell cycle analysis was performed by flow cytometry in HT-29 cells treated with formulations F1 (MD:CGA, 2:1) and F3 (AG:CGA, 2:1). Systems based on MD, AG, and S yielded spherical, smooth particles with an average diameter of about 360 nm, whereas those based on CMC and HPMC produced smaller particles of about 251 nm, likely due to lower polymer concentrations. Unloaded NPs exhibited low overall cytotoxicity in both SW480 and HT-29 CRC cell lines, confirming their biocompatibility. In contrast, CGA-loaded formulations induced a greater reduction in cell viability and proliferation, particularly in HT-29 cells. Cell cycle analysis revealed a slight increase in the sub-G1 population, with F1 promoting S-phase accumulation and F3 causing G2/M arrest. Among all formulations, those containing AG demonstrated significantly enhanced anticancer activity in CRC cells compared with free CGA. This effect appears to be cytostatic rather than apoptotic, suggesting a promising direction for further exploration.