Characterizing the leaf-level physiological responses to biotic stress may provide a basis for improving the early detection of mite pests, particularly those that are difficult to detect through conventional methods. The aim of this study was therefore to evaluate the impact of Dolichotetranychus floridanus (Banks) on the daily dynamics and magnitude maximum photochemical efficiency of PSII (Fv/Fm) and leaf temperature in Ananas comosus (L.) Merr. over a 24-hour cycle, investigating whether mite infestation produces detectable changes in these variables. In ten pineapple plants (var. Pérola), the maximum photochemical efficiency of PSII (Fv/Fm) was determined by pulse-modulated fluorometry after dark adaptation, and leaf temperature was recorded using infrared thermography, at two-hour intervals over a 24-hour cycle. Plants were categorized into three infestation levels based on mite count per plant: low (<10), medium (10–40), and high (>40 mites). For leaf temperature analysis, plants were classified simply as infested or non-infested. Linear mixed models (LMM) were used to analyze daily thermal and Fv/Fm dynamics, incorporating time (polynomial or cyclic structure) and mite abundance as fixed effects, with plant as a random effect and an AR(1) temporal autocorrelation structure. Additionally, differences in maximum, minimum, and mean leaf temperatures were evaluated using the Kruskal-Wallis test (a = 0.05). Post-hoc comparisons were performed using the Compact Letter Display (CLD) method, and all data visualization was conducted using the ggplot2 package in R (R Core Team 2025). Results showed that 24-hour dynamics were dominated by the temporal component (p < 0.001), featuring a quadratic thermal pattern and circadian oscillation in Fv/Fm. Mite’s infestation did not significantly alter the shape or amplitude of these cycles; however, infested plants tended to show higher leaf temperatures across all thermal parameters. The Kruskal-Wallis test confirmed that infested plants maintained significantly higher temperatures than non-infested plants, with median maximum (21.70°C vs. 21.25°C), minimum (21.40°C vs. 20.65°C), and mean temperatures (21.60°C vs. 20.90°C) (p < 0.05). Although these differences were modest in absolute terms (0.45–0.75°C), they may be physiologically relevant, though its biological and practical importance at the crop level remains to be evaluated. These findings offer preliminary leaf-level evidence that could inform future investigation at broader spatial scales.