<p>Non-native species threaten freshwater biodiversity and ecosystem services (ES), which are crucial for environmental health and hold significant socioeconomic and cultural value. This study examines the perceived impacts of the signal crayfish (<i>Pacifastacus leniusculus</i>) in Montesinho Natural Park, Portugal, combining scientific data with local ecological knowledge (LEK) across six rivers. Face-to-face interviews with 110 residents (balanced genders) revealed that 46% of men and 26% of women reported sightings of the crayfish, with introductions perceived around 2015 and 2017, respectively. Most respondents (79%) believed populations had significantly increased, which aligns with our scientific data. Participants rated the crayfish’s ES impact on a 0–5 scale, with the highest perceived effects on food sources for other species (4.7) and habitat provision (4.4), followed by water quality (3.7). Erosion (1.7) and pest regulation (3.0) were perceived as the least affected services. Both genders identified fish, especially the brown trout (<i>Salmo trutta</i>), as the most affected taxonomic group (87.5% men, 77.8% women). Our findings highlight not only the importance of community engagement and monitoring in managing biological invasions, but also the value of gender-based perceptions to reconstruct invasion history and better understand the ecological and social impacts of signal crayfish.</p>