Rapidly assessing biodiversity is essential for environmental monitoring; however, traditional approaches are limited in the scope needed for most ecological systems. Environmental DNA (eDNA) based assessment offers enhanced scope for assessing biodiversity, while also increasing sampling efficiency and reducing processing time, compared to traditional methods. Here we investigated the effects of landuse and seasonality on headwater community richness and functional diversity, via spatio-temporal dynamics, using both eDNA and traditional sampling. We found that eDNA provided greater resolution in assessing biodiversity dynamics in time and space, compared to traditional sampling. Community richness was seasonally linked, peaking in spring and summer, with temporal turnover having a greater effect on community composition compared to localized nestedness. Overall, our assessment of ecosystem function shows that community formation is driven by regional resource availability, implying regional management requirements should be considered. Our findings show that eDNA based ecological assessment is a powerful, rapid and effective assessment strategy that enables complex spatio-temporal studies of community diversity and ecosystem function, previously infeasible using traditional methods.