With their accession to the EU in 2004, Poland and Hungary had to implement the Natura 2000 programme for biodiversity protection. In both countries NGOs have been active throughout the implementation process. Forms and outcomes of NGO involvement, however, differed. Hungarian NGOs were very influential during the site designation phase, working closely with the governmental authorities and contributing considerably to the country’s site proposal. In Poland, the form of NGO’s involvement changed from opposition towards the government (publication of a Natura 2000 shadow list) to close cooperation with public institutions, resulting in a significant expansion of site designations. This paper analyses the role and impact of NGOs on Natura 2000 implementation in Poland and Hungary and seeks explanation for the observed differences with reference to the theoretical background of policy networks and advocacy coalitions. The qualitative data used for the study is based on in-depth interviews with NGOs representatives and officials of public institutions engaged in Natura 2000 implementation. The comparison shows that the existing architecture of the sector of biodiversity governance is decisive for NGO activities and determines their role and impact. In the European multilevel governance setting new formal and informal opportunities were given to NGOs. In both countries NGOs became stronger during the Natura 2000 process. We argue that this was a result of the establishment of multi-level policy networks between the European Commission, NGOs and public institutions, based on resource dependencies. These networks were powerful enough to overcome dominating policy patterns in both countries. The differences found between Hungary and Poland could be explained by different discoursive positions of the responsible ministries. The change of government in Poland in 2007 shifted the discourse towards supporting conservation, which enabled the formation of an advocacy coalition between the government and NGOs.