Outbreaks of infectious viruses resulting from spillover events from bats have brought much attention to bat-borne zoonoses, which has motivated increased ecological and epidemiological studies on bat populations. Field sampling methods often collect pooled samples of bat excreta from plastic sheets placed under-roosts. However, positive bias is introduced because multiple individuals may contribute to pooled samples, making studies of viral dynamics difficult. Here, we explore the general issue of bias in spatial sample pooling using Hendra virus in Australian bats as a case study. We assessed the accuracy of different under-roost sampling designs using generalized additive models and field data from individually captured bats and pooled urine samples. We then used theoretical simulation models of bat density and under-roost sampling to understand the mechanistic drivers of bias. The most commonly used sampling design estimated viral prevalence 3.2 times higher than individual-level data, with positive bias 5–7 times higher than other designs due to spatial autocorrelation among sampling sheets and clustering of bats in roosts. Simulation results indicate using a stratified random design to collect 30–40 pooled urine samples from 80 to 100 sheets, each with an area of 0.75–1 m2, and would allow estimation of true prevalence with minimum sampling bias and false negatives. These results show that widely used under-roost sampling techniques are highly sensitive to viral presence, but lack specificity, providing limited information regarding viral dynamics. Improved estimation of true prevalence can be attained with minor changes to existing designs such as reducing sheet size, increasing sheet number, and spreading sheets out within the roost area. Our findings provide insight into how spatial sample pooling is vulnerable to bias for a wide range of systems in disease ecology, where optimal sampling design is influenced by pathogen prevalence, host population density, and patterns of aggregation.