An agro-environmental payment for the management of the so-called ‘Biodiversity Promotion Areas’ (BPA) has been used to accomplish biodiversity conservation goals in Switzerland. These areas have been managed according to specific limitations on mowing dates and fertilizers. We assessed the regional-scale effectiveness of BPA implementation within Ticino Canton by answering the following questions: (i) is plant species diversity higher in BPA than in conventionally managed grasslands (CMG)? (ii) which are the differences between BPA and CMG in terms of climatic, topographical, ecological, and vegetation variables? (iii) which vegetation types, functional groups, and plant species are specifically related to BPA? A total of 242 vegetation surveys (64 in BPA and 178 in CMG, respectively) was carried out in 55 farms and the main climatic and topographic features were assessed. Differences in terms of plant diversity, ecological indicator and pastoral values, species functional groups, vegetation types, and indicator species between BPA and CMG were assessed. The BPA harboured a higher plant diversity. They were located in steeper areas, at higher elevations, and characterised by lower soil nutrient content, mowing tolerance, and pastoral value than CMG. Dry meadow species number and cover were higher in BPA, while nutrient-rich meadow species number was higher in CMG. The species associated to BPA belonged to a wider range of functional groups and 38% of them belonged to the national list for biodiversity promotion in agriculture, whereas no species associated to CMG belonged to that list. Thus, our results confirmed the effectiveness of BPA for biodiversity conservation for the Southern Swiss Alps.