There is a growing concern about food security and sustainability of agricultural production in developing countries. However, there are limited attempts to quantify agro-biodiversity losses and relate these losses to soil degradation and crop productivity, particularly in Tigray, Ethiopia. In this study, spatial variation in agro-biodiversity and soil degradation was assessed in 2000 and 2005 at 151 farms in relation to farm, productivity, wealth, social, developmental and topographic characteristics in Tigray, northern Ethiopia. A significant decrease in agro-biodiversity was documented between 2000 and 2005, mainly associated with inorganic fertilizer use, number of credit sources and proximity to towns and major roads. Agro-biodiversity was higher at farms with higher soil fertility (available P and total N) and higher productivity (total caloric crop yield). Low soil organic matter, few crop selection criteria and steep slopes contributed to soil erosion. Sparsely and intensively cultivated land use types, as determined from satellite images, were associated with high and low agro-biodiversity classes, respectively, as determined during on-farm surveys in 2005. This study gives insight into the recent changes in and current status of agro-biodiversity and soil degradation at different spatial scales, which can help to improve food security through the maintenance of agro-biodiversity resources.