As human activities drive biodiversity decline, effective biomonitoring is more crucial than ever to track species distribution changes and inform conservation and restoration actions. Environmental DNA (eDNA) metabarcoding has emerged as a promising tool for the simultaneous detection of multiple taxa. However, while substrates play a crucial role in eDNA studies, limited research has compared substrate performance for terrestrial vertebrate detection, leaving a critical gap in empirical knowledge for large-scale application. This study evaluates and compares the effectiveness of three easy-to-collect substrates: soil, leaf swabs and spider webs, for broad terrestrial vertebrate eDNA monitoring. Specifically, we examined taxonomic richness overlaps among substrates, their effects on wild vertebrate detection probabilities and within-sample PCR repeatability. We analyzed 120 samples from the Landes Forest, an intensively managed temperate forest in Western France, and included additional samples from the Montpellier zoo to validate our detection capabilities. Using metabarcoding with 12Sv5 and 16Smam primers, we identified 67 taxa at the genus or species level. Our results demonstrate that spider webs consistently outperformed the other substrates, followed by leaf swabs and soil. These findings highlight the advantages of airborne-derived substrates (leaf swabs and spider webs) over soil and position spider webs as optimal for maximizing detection probabilities in rapid eDNA surveys, emphasizing their potential for efficient, scalable biomonitoring. Further research is needed to identify factors affecting eDNA detectability from these substrates, aiming to standardize procedures and move from proof-of-concept to broad use by researchers and managers.