This study presents the development of melatonin-coated lactoferrin-chitosan nanoparticles (ETP-CS-LF-MLT-NPs) using ionic gelation and carbodiimide coupling for colorectal cancer treatment. The nanoparticles were characterized by an average size of 208.7 ± 1.25 nm, a zeta potential of 30.77 ± 1.21 mV, and 82.45 % drug encapsulation efficiency. In vitro drug release studies showed sustained, pH-responsive release, with 98.68 ± 4.12 % released at pH 5.5 over 24 h. The nanoparticles exhibited significant cytotoxicity in HCT116 cells (IC50 = 15.32 μg/mL), inducing ROS generation, apoptosis, and G2/M cell cycle arrest, with notable downregulation of BCL2 gene expression. Enhanced cellular uptake due to lactoferrin targeting improved therapeutic efficacy. In In vivo studies, the nanoparticles demonstrated significant tumor reduction and selective colon accumulation in a DMH-induced colorectal cancer rat model, along with improved pharmacokinetics, showing extended plasma circulation and bioavailability compared to free etoposide. Biocompatibility assays, including hemolysis (<1 %), platelet aggregation, and HET-CAM tests, confirmed the safety profiling of the prepared nanoparticles. The nanoparticles also inhibited Proteus mirabilis (ZOI = 1.9 cm) and exhibited promising effects on the gut microbiome of treated animals. Altogether, ETP-CS-LF-MLT-NPs hold great potential for targeted colorectal cancer therapy, improving drug delivery, tumor targeting, bioavailability, and reducing systemic toxicity.