In this investigation, the incidence and intensity of gastrointestinal parasites of domestic pigeons were evaluated, additionally, in vitro and in vivo evaluation of the antiparasitic activity of chitosan nanoparticles against the most predominant gut parasite. Therefore, 240 domestic pigeons (160 adults and 80 squabs) obtained from different localities in Giza governorate, Egypt, from February to July 2021, were subjected to parasitological and postmortem examination. The results revealed that 97% of pigeons were vulnerable to single or mixed gastrointestinal parasites. The detected helminths were identified as Capillaria columbae (C. columbae) with a total incidence of (12.5%), Ascaridia columbae (A. columbae) (83.3.%), Heterakis gallinarum (H. gallinarum) (18.7%), Raillietina cesticillus (R. cesticillus) (7.5%), Raillietina echinobothrida (R. echinobothrida) (29%), Choanotaenia infundibulum (C. infundibulum) (22.9%), Davainea proglottina (D. proglottina) (26.6%), and Cotugnia proglottina (C. proglottina) (14.5%). At the same time, the identified protozoan parasites were Trichomonas gallinae (T. gallinae), and Eimeria columbae (E. columbae), with a total incidence of 25 and 79%, respectively. Helminths and Eimeria infections were higher in adults than squabs, while T. gallinae infection was reported with a higher incidence in squabs (62.5%) than adults (6.2%). From our findings, A. columbae was the most predominant gut parasite in the examined pigeons. Thus, it was subjected to in vitro and in vivo treatment with chitosan nanoparticles. Serum and tissue samples were collected from the birds which have been used in the in vitro study to evaluate the oxidative stress markers as malondialdehyde (MDA), Nitric oxide levels and Tumor Necrosis Factor-alpha (TNF-α) and Interleukin-1β activity also, chitosan nanoparticles- treated worm ultrastructure were determined using scanning electron microscopy (SEM). Finally, we recommend periodic monitoring of pigeon's farm to detect the parasitic infestation, and from our results, we recommend chitosan nanoparticles as a potent nematocidal agent.