Climate change and increasing extreme weather intensify pest damage and threaten agricultural ecosystems. Although pest management still relies heavily on pesticides, biological control is crucial for sustainable agriculture. However, how temperature influences the behaviour and predation strategies of natural enemies remains poorly understood. Therefore, we aimed to investigate temperature-dependent changes in locomotor and predatory behaviour to better understand predator–prey dynamics and the ecological adaptability of natural enemies under future climate conditions. We examined the behavioural responses of the predatory mite Amblyseius andersoni to two pest species, the western flower thrips Frankliniella occidentalis and the two-spotted spider mite Tetranychus urticae, across five temperatures (15, 20, 25, 30, and 35°C) after 30 min of acclimation, focusing on locomotion (speed, area explored, proportion of time moving, encounter rate) and predation (handling time, predation rate, short- term preference). In addition, nutritional value of the two prey species was inferred from daily predation and oviposition rates. Each experiment was replicated 20 times at each temperature. The results showed that both temperature and acclimation time significantly influenced locomotor activity. A. andersoni exhibited peak movement speed, area explored, and proportion of time moving at 30°C; however, these metrics were significantly lower at 35°C. Across all temperatures, F. occidentalis consistently moved faster and explored a larger area than T. urticae. Furthermore, the handling time was shorter at higher temperatures for both prey, and the time gap between prey types narrowed. At 20°C and 30°C, prey preference showed opposite patterns: preference for F. occidentalispeaked at 30°C, while preference for T. urticae peaked at 20°C. In addition, the nutritional value of the two pest species was inferred from daily predation and oviposition rates. Although daily consumption of T. urticaewas higher, the oviposition rates were comparable whether A. andersoniconsume F. occidentalis or T. urticae, indicating that F. occidentalis are more nutritious and energy-rich than T. urticae. All results indicate that temperature and acclimation time strongly affected locomotior activity and predation behaviour. Temperature-dependent differences in locomotion and handling time, together with differences in nutritional value, likely underlie the observed shifts in predation rate and prey preference in A. andersoni. Overall, these findings demonstrate that temperature plays a key role in shaping the locomotor activity and predatory behaviour of polyphagous natural enemies under multi-prey conditions.