In this study, chitosan nanoparticles (ChNs) were used as an adsorbent for single and simultaneous uptake of cationic (methylene blue (MB)) and anionic (methyl orange (MO)) dyes. ChNs were prepared based on the ionic gelation method using sodium tripolyphosphate (TPP) and characterized by zetasizer, FTIR, BET, SEM, XRD, and pHPZC. The studied parameters that affect removal efficiency included pH, time, and dyes' concentration. The results showed that in single-adsorption mode, the removal of MB is better in alkaline pH, contrary to MO uptake which presents higher removal efficiency in acidic media. The simultaneous removal of MB and MO from the mixture solution by ChNs could be achieved under neutral conditions. The adsorption kinetic results showed that adsorption of MB and MO for both single-adsorption and binary adsorption systems comply with the pseudo-second-order model. Langmuir, Freundlich, and Redlich-Peterson isotherms were used for the mathematical description of single-adsorption equilibrium, while non-modified Langmuir and extended Freundlich isotherms were used to fit the co-adsorption equilibrium results. The maximum adsorption capacities of MB and MO in a single dye adsorption system were 315.01 and 257.05 mg/g for MB and MO, respectively. On the other hand, and for binary adsorption system, the adsorption capacities were 49.05 and 137.03 mg/g, respectively. The adsorption capacity of MB decreases in solution containing MO and vice versa, suggesting an antagonistic behavior of MB and MO on ChNs. Overall, ChNs could be a candidate for single and binary removal of MB and MO in dye-containing wastewater.