In Niokolo Koba National Park (NKNP), in the South East of Senegal, we studied the current spotted hyena (Crocuta crocuta) population size, density and distribution and assessed human-large carnivores’ conflicts. We implemented 26 calling stations operations, installed 87 camera traps and obtained data from tourists, local guides and rangers. In addition, a questionnaire survey was carried out in 31 villages around the park. Our results showed an estimated density from 1.77 to 3.53 spotted hyenas/100 km² depending on used response rates (50–100%) with a minimum population size between 144 and 288 individuals. According to the query survey, the spotted hyena is responsible for the majority of livestock predation around the park (87% of all attacks on livestock), followed by African wild dog (6%), lion (4%) and leopard (3%). A total of 63.1% of all attacks were on cattle, while sheep and goats represent the remaining 36.9%. Spotted hyena and lion mainly predated on livestock at night (93.5% and 71.4% of all attacks, respectively) in contrast to predation by African wild dog and leopard, for which the majority of the attacks occurred during the day (63.9% and 62.5%, respectively). Furthermore, there was more livestock killed during the wet season (58% of all attacks) than during the dry season (42% of all attacks). The distribution of conflicts across five sectors surrounding the park illustrate that the West sector is most impacted (49% of all attacks; districts of Linkering and Medina Gounass) followed by the North West (32%; districts of Dialocoto and Missira). Insights into the severity and dynamics of human-wildlife conflict will contribute towards finding effective mitigation measures, minimising loss of livestock and reducing conflicts.