Aquatic organisms in pharmacology and toxicology research are often exposed to compounds in isolation prior to physiological or behavioural testing. Recent evidence suggests that the presence of conspecifics during a stressful event can modulate behavioural outcomes (called ‘social buffering’) when testing occurs within the same context. It is unknown, however, whether the social environment during exposure interacts with the efficacy of anxiety-altering substances when subsequently tested in the absence of conspecifics. In this study, zebrafish were individually exposed to habitat water or ethanol (1.0% vol/vol) while untreated conspecifics were visually present or absent during dosing. Using the novel object approach test, a validated test of boldness and anxiety-like behaviour, we observed significantly greater effects of ethanol in isolated fish, compared to fish with a view of conspecifics during dosing. These results were not explained by altered locomotion during exposure, which might otherwise increase drug uptake. This highlights the need to consider the social environment during exposure when conducting and interpreting behavioural research involving drug or toxicant exposure.