Alpha-mangostin (M) and resveratrol (R), dual-drugs-loaded mucoadhesive thiolated chitosan-based nanoparticles (NPs) coated by Eudragit® S100 (S) were developed for colon-specific delivery and synergistic activity against colon cancer cells. The NPs were prepared by the ionotropic gelation method and coated with S. The particle size and zeta potential of NPs before and after the coating process were observed. The M and R loading efficiency, mucoadhesive properties, as well as release patterns were examined. Moreover, the activity against colon cancer cells of M, R, and NPs were studied for their synergistic activity. M and R-loaded NPs (MR-TNPs) were spherical in shape with sizes of around 540 nm and zeta potential of +39 mV. The S coating of MR-TNPs provided larger particle sizes which offered lower zeta potential. However, it created an increase in M and R loading, prevented M and R release at the upper gastrointestinal tract, and enhanced M and R reaching the colon. S dissolved at pH > 7.0 while thiolated chitosan formed the mucoadhesion, resulting in M and R remaining in the colon and allowing them to enter the colon cancer cells. The half-maximal inhibitory concentration values of NPs was dramatically decreased when M and R were dually loaded into the NPs, which indicated significantly higher activity against colon cancer cells. Moreover, M and R loading at this ratio applied synergistic efficiency. The results illustrated that NPs successfully loaded drugs and achieved synergistic efficiency. This system could be promising in facilitating targeted nanomedicines for the treatment of colon cancer.