Purpose: To investigate the effect of fixed combination versus concomitant unfixed topical glaucoma treatment on patients’ adherence and ocular surface health. Patients and Methods: This is a 6-month, prospective, parallel-group, observational study in patients with ocular hypertension (OHT), primary open-angle glaucoma (POAG), or exfoliation glaucoma (XFG). A total of 142 patients with similar baseline characteristics were enrolled in this study. Seventy-one patients received a Latanoprost 0.005%/Timolol 0.5% fixed combination in the evening, whereas seventy-one patients received the unfixed treatment with Latanoprost 0.005% in the evening and Timolol 0.5% twice daily. The primary outcome was the adherence rate at baseline, and at the 3- and 6-month follow-up visits. The secondary outcomes included the signs of ocular surface disease (OSD) and intraocular pressure (IOP). Results: The adherence of patients treated with the fixed combination was higher than the unfixed treatment at the 3-month (78.0% vs. 63.0%,p< 0.001) and at the 6-month visits (73.0% vs. 58.5%,p< 0.01). The Break-up Time, Schirmer test, and Van Bijsterveld score were worse in the unfixed group at baseline and all subsequent examinations (p< 0.05 for all comparisons). There were no differences in the mean IOP between groups at baseline, 1-, and 3-month visits. IOP appears higher in the unfixed group at 6 months (16.7 vs. 15.0 mmHg,p< 0.01). Conclusion: The patients with ocular hypertension and primary open-angle glaucoma treated with a fixed combination are significantly more adherent and show a healthier ocular surface than those treated with an unfixed combination. The study provides significant evidence of the benefits of fixed combination treatment.