In this work, thermal imaging is employed to study the opto-thermal response of apples (Malus domestica Borkh.), assessing their post-harvest evolution through the estimation of thermal diffusivity. A non-destructive experimental procedure based on mid-wave infrared (MWIR) thermal camera (3–5 µm) and localized heating with a visible laser is developed, enabling spatially and temporally resolved surface temperature measurements. Temperature fields are recorded at different time points and radial distances from the heated spot. A theoretical model based on Fourier thermal diffusion equation is formulated to describe the spatio-temporal evolution of surface temperature. After validation on a reference sample, the method is applied to Golden and Red Delicious apples over a 28-day storage period at room temperature. Red Delicious apple exhibits higher mean diffusivity values without significant temporal changes, whereas a progressive increase in diffusivity is observed for Golden Delicious apples. These results show that thermal diffusivity is sensitive to post-harvest physiological changes in apple tissue and may be associated with intrinsic properties such as tissue density and water content. By relating laser-induced temperature fields to the estimation of thermal diffusivity, this approach enables the non-destructive, quantitative assessment of thermal diffusivity, showing potential for fruit maturity and quality assessment, which are of high importance in agri-food monitoring applications.