This study evaluated the short-term response to pour-on eprinomectin (EPM) treatment in buffaloes naturally infested by Haematopinus tuberculatus, focusing on haematobiochemical parameters, serum and milk cortisol levels, and milk production. Twenty-four Italian Mediterranean buffaloes were allocated to two groups: an untreated Control group and a group receiving 0.5 mg EPM/kg body weight. From treatment day (T0), blood was sampled every 14 days, while milk yield was recorded and milk samples collected every 7 days for 4 weeks. Haematobiochemical parameters, serum and milk cortisol, and milk composition were analysed. Data were statistically analysed using a linear mixed-effects model including treatment, time (as fixed categorical effects) and their interaction, T0 as a covariate and the animal as a random effect. No differences were detected for cortisol and most haematobiochemical parameters between groups (p > 0.05). Haptoglobin showed lower values in EPM-treated buffaloes when compared to Controls (34.8 vs. 43.6 μg/mL; p = 0.026). Milk yield remained unaffected by treatment (p > 0.05), whereas milk fat was higher (83.1 vs. 69.1 g/kg; p = 0.011) and milk urea was lower (36.1 vs. 40.1 mg/dL; p = 0.002) in EPM-treated buffaloes. Overall, EPM treatment against H. tuberculatus infestation was associated with limited short-term changes in physiological and productive responses in lactating buffaloes, including lower serum haptoglobin concentrations and differences in selected milk composition traits.