<p>Animals exhibit behavioural responses to environmental stimuli. To date, various experimental assays have been conducted to investigate the molecular and neural mechanisms underlying behavioural responses in <i>Caenorhabditis elegans</i>. However, behavioural outcomes can vary even under the same stimulus due to factors such as past experience, individual circumstances, and modifications in behavioural assay procedures, which complicate the interpretation of results. In this study, we focussed on the composition of the behavioural assay media and compared the behaviours of <i>C. elegans</i> in response to temperature and odour stimuli using three commonly used media: nematode growth medium (NGM), thermotaxis (TTX), and chemotaxis (Chemo) assay media. Our results demonstrated that in thermotaxis, <i>C. elegans</i> exhibited stable behavioural decisions across all examined media. However, the population of worms reaching the preferable areas was smaller in Chemo and TTX media compared with NGM due to prolonged exploration period and reduced migration speed. By contrast, in chemotaxis, we did not observe significant differences across the tested media. This study provides insights into the significant effects on <i>C. elegans</i> behaviour caused by the modifications of the behavioural assay media and emphasises the importance of further studies to explore the detailed neural mechanisms that regulate animal behaviour under diverse environmental factors.</p>