Flying-foxes (genus Pteropus) are the natural host of Hendra virus (HeV) which periodically causes fatal disease in horses and humans in Australia. The presence of flying-foxes in urban areas has increased in recent decades, and often provokes negative community sentiments resulting in calls for the dispersal of urban flying-fox roosts. However, it has been hypothesised that disturbance of urban roosts may result in a stress-mediated increase in virus excretion, promoting infection in flying-foxes, and increasing the risk of spillover to horses and consequently humans. We sought to examine the impact of dispersal on urinary HeV infection and cortisol dynamics in flying-foxes. The data were analysed in generalised linear mixed models using restricted maximum likelihood. The difference in mean HeV prevalence in samples collected before (4.9%), during (4.7%) and after (3.4%) roost disturbance was non-significant (P = 0.440). Similarly, the difference in mean urinary cortisol concentrations was non-significant (before = 22.71 ng/mL, during = 27.17, after = 18.39) (P= 0.550). We did find associations between cortisol concentration, season and region, while the effect of dispersal on cortisol concentration approached statistical significance for region, plausibly reflecting the nature and timing of disturbance. We also found a positive statistical association between HeV excretion status and urinary cortisol concentration, but elaborating any causal association was beyond the scope of the study. These findings usefully inform public discussion and policy development in relation to Hendra virus and flying-fox management. Qualitative assessment of behavioural distress associated with roost disturbance showed that the severity of impact reflected the nature and timing of the activity, and highlights the need for a ‘best practice’ approach to dispersal. While the mobility of flying-foxes provides some capacity to escape anthropogenic disturbance, their increasing urban presence may subject them to chronic roost disturbance and harassment, the consequences of which are unknown.