To counteract the ongoing biodiversity loss due to intensified agricultural practices, agri-environmental schemes have been introduced in most European countries. A systematic monitoring is needed to optimise their effectiveness in promoting biodiversity. New and automated methods, such as passive acoustic monitoring (PAM), offer a promising alternative to traditional, labour-intensive, and costly methods. However, we know relatively little about whether differences in biodiversity due to land use can be related to differences in acoustic indices, and whether spatio-temporal factors are modifying the relationship. To assess the potential of PAM as an alternative to traditional methods for evaluating the effectiveness of agri-environmental measures, we explored whether bird and grasshopper species richness and insect abundance as grassland diversity indicators are related to the acoustic complexity index and to land use at local scales (e.g., fertilisation), and landscape scales (e.g., percentage of woody structure). We also examined whether these relationships depend on the peak vocalisation activity of the indicator group (morning vs. midday). Local vegetation structure increased insect abundance, which was related to an increased acoustic complexity index at midday. A higher percentage of agri-environmental schemes in the landscape increased bird species diversity, which was related to an increased acoustic complexity index in the morning. Passive acoustic methods to monitor biodiversity and evaluate the effectiveness of conservation measures are a promising tool. This study demonstrates that the acoustic complexity index is able to indirectly show changes in land-use via species richness or abundance. Furthermore we would like to point out that the suitability depends on the indicator group and its activity patterns.