Background: Ocular surface frequently affects glaucoma patients. In this paper we aimed at evaluating the impact of glaucoma surgery on the ocular surface of patients who received unilateral trabeculectomy. Methods: 26 consecutive patients successfully treated with trabeculectomy on one eye (Trab Eye) and under control with topical treatments on the fellow eye (Med Eye) were included in this observational study. They received IDEEL and OSDI questionnaires, Tear Film Osmolarity (TFO), grading of conjunctival hyperemia, fluorescein tear break-up time (tBUT), grading of corneal staining and Schirmer test. Results: IDEEL and OSDI scores were 48 ± 38 and 11 ± 12, respectively, with moderate correlation (r = 0.50, p = 0.03). Compared with Med eyes, Trab Eyes had higher tBUT (6.5 ± 3.5 vs. 5.1 ± 2.7 s, p = 0.004), lower conjunctival hyperemia (0.8 ± 0.9 and 1.7 ± 1.1 respectively, p < 0.001) and lower corneal staining (0.3 ± 0.5 and 0.6 ± 0.5, respectively, p = 0.03). Correlation between corneal staining and conjunctival hyperemia was 0.55 in Trab Eyes (p = 0.01) and 0.44 in Med Eyes (p > 0.05). Patients with bilateral corneal staining had had threefold worse questionnaire scores (p < 0.05). The duration of treatment and the daily exposure to preservatives did not directly affect OS parameters in this cohort of patients. Conclusions: Patients receiving successful trabeculectomy showed better OS homeostasis (higher TBUT, lower grading of conjunctival hyperemia and corneal staining) than fellow medically treated eyes. Presence of corneal epithelial damage in both eyes is the factor more consistently affecting questionnaire scores.