Campylobacteriosis is a significant zoonotic disease with substantial public health and economic impacts. This study aimed to investigate the prevalence of Campylobacter spp. in animals and humans in the New Valley Governorate, Egypt, and identify potential associated risk factors. A total of 432 samples were collected, including 320 animal samples (rectal swabs (207) and milk (113)) and 112 human samples (stool (66) and hand swabs (46)). Isolation and identification of Campylobacter spp. were performed using conventional cultural and biochemical methods. Out of all examined samples, the overall prevalence of Campylobacter spp. was 32.9% in animals and 74.2% in humans. Sheep exhibited the highest prevalence (38.7%), followed by goats (27.4%), and cattle (24.3%). The most important associated risk factors for Campylobacteriosis are locality, age, sex, and health status of animals and humans. The high prevalence of Campylobacter spp. in both animal and human samples reveals that animal feces act as a serious source of Campylobacteriosis and alarm the circulation of Campylobacter spp. between animals and humans as an important zoonotic pathogen.