The Antillean manatee in Guatemala faces multiple threats resulting from habitat loss and human interactions such as motorboat collisions and poaching. While some conservation efforts have been initiated in Río Dulce National Park (RDNP), there is a lack of studies examining the current perceptions of threats to the species. This knowledge gap may hinder the development of effective conservation strategies that consider both the species and local communities. Our objective was to analyze the perceptions of key local stakeholders concerning the Antillean manatee, including its distribution, threats, attention to strandings, and the current status of conservation in the protected area of RDNP. From July to October 2019, we conducted 50 semi-structured interviews with technicians and river users, such as fishermen and boat captains. Additionally, we held two workshops: one aimed at developing a participatory mapping exercise and another to validate the information. Each workshop was divided into two groups: one for technicians and another for river users. A total of 68 people participated in the workshops, the majority of respondents were male (n = 47), ranging in age from 19 to 71, and predominantly local to the area. Their activities in the river included fishing, biological monitoring, transportation, and providing tourism services. Perceived threats to the Antillean manatee included motorboat collisions, poaching, bycatch, habitat degradation, and water pollution. The focus groups also identified key observations, such as mother-calf sightings, specific areas of risk for manatee-boat collisions, areas with a higher presence of aquatic vegetation, and additional threats such as habitat degradation and poaching. Our results demonstrate that stakeholders possess a fundamental understanding of the manatee, its threats, and its distribution. They also highlight a noticeable lack of knowledge, specifically regarding the attention given to manatee strandings. It is recommended that conservation strategies include environmental education adapted to local conditions, as well as the promotion of manatee studies and their dissemination at the local level, including attention to strandings in the area.