Rising temperatures and increasingly frequent soil droughts are reshaping forest ecosystems, driving shifts in species composition. These shifts may alter canopy structure and microclimate, with potential feedbacks on tree physiological responses and net carbon uptake under warming. Yet, their effects remain poorly understood. We investigated how species composition influences canopy size, microclimate, key foliar functional traits, and photosynthetic and respiratory thermal acclimation in mature European beech (Fagus sylvatica L.) and downy oak (Quercus pubescens Willd.) across a beech-oak ecotone in Switzerland. Canopy structure and microclimate differed markedly between pure and mixed forest stands, with beech exhibiting larger canopies and air cooling in the pure stand, whereas downy oak showed the opposite trend. These differences had little impact on photosynthetic and respiratory thermal acclimation, but led to species-specific adjustments in leaf traits in both species. Variation in respiration rates across sites and canopy levels was strongly associated with specific leaf area, suggesting that morphological adjustments play a greater role than physiological acclimation in regulating carbon exchange at this ecotone. Further research should explore how tree-level structural adjustments and local environmental drivers modulate beech and oak foliar functional and physiological responses in mature forests to better predict their vulnerability to future climate.