Context Road infrastructure construction is integral to economic development, but negatively affects biodiversity. To mitigate the negative impacts of infrastructure, various types of wildlife crossings are realized worldwide, but little is known about their effectiveness, and cost-effectiveness. Objective The paper contributes to the methodological and empirical discussion on the effectiveness of wildlife crossings for enhancing the quality of surrounding nature and its cost-effectiveness by analyzing a large-scale wildlife-crossings program in the Netherlands. Method A multi-criteria cost-benefit analysis is applied, comprised of monetary and non-monetary measures, and a mixed-method approach is used to determine ecological effects. Ecological effects are expressed in the standardized weighted hectare measurement of threat-weighted ecological quality area (1 T-EQA = 1 ha of 100% ecological quality, averagely threatened). Cost-effectiveness is calculated comparing the monetary costs of intervention with ecological benefits (Euro costs/T-EQA), for different types of wildlife crossings and for two other nature policies. Results The Dutch habitat defragmentation program has induced an increase in nature value of 1734 T-EQA at a cost of Euro 283 million. Ecological gains per hierarchically ordered groups of measures differ strongly: The most effective are ecoducts (wildlife crossing bridges) followed by shared-use viaducts and large fauna tunnels. Ecoducts generated the largest gain in nature value, but were also the most costly measures. In terms of cost-effectiveness, both large fauna tunnels and shared-use viaducts for traffic and animals outperformed ecoducts. Conclusions Ecoducts deliver ecologically, but their cost-effectiveness appears modest. Purchasing agricultural land for restoration of nature appears more cost-effective than building wildlife crossings. Yet, reducing environmental pressures or their effects on existing nature areas is likely to be most cost-effective.