The effects of preservatives of antiglaucoma medications on corneal surface and tear function have been widely shown in literature; it’s not the same as regards the active compounds themselves. The purpose of our study was to compare Ocular Surface Disease (OSD) signs and symptoms of Tafluprost 0.0015% versus preservative free (PF) Timolol 0.1% eyedrops in ocular hypertensive (OH) and in primary open-angle glaucoma (POAG) patients. A cross-sectional study included patients in monotherapy for at least 36 months with Tafluprost 0.0015% (27) or PF Timolol 0.1% (24) and 20 healthy age and sex-matched volunteers. All subjects underwent clinical tests (Schirmer I and break-up time), in vivo confocal microscopy (IVCM) and were surveyed using Ocular Surface Disease Index (OSDI) and Glaucoma... Symptoms Scale (GSS) questionnaires. The groups were compared with ANOVA, Kruskal-Wallis test, t-test, Mann-Whitney test and Bonferroni’s adjustment of p-values. No significant differences were found in questionnaires scores, clinical tests, IVCM variables between therapy groups. Tafluprost 0.0015% group showed significantly higher OSDI score, basal epithelial cells density, stromal reflectivity, sub-basal nerves tortuosity (p = 0.0000, 0.037, 0.006, 0.0000) and less GSS score, number of sub-basal nerves (p = 0.0000, 0.037) than controls but similar clinical tests results (p > 0.05). PF Timolol group had significantly higher OSDI score, basal epithelial cells density, stromal reflectivity and sub-basal nerve tortuosity (p = 0.000, 0.014, 0.008, 0.002), less GSS score, BUT and number of sub-basal nerves (p = 0.0000, 0.026, 0.003) than controls. Compared to PF Timolol 0.1%, Tafluprost 0.0015% showed similar safety with regards to tear function and corneal status and a similar tolerability profile. Both therapy groups show some alterations in corneal microstructure but no side effects on tear function except for an increased tear instability in PF Timolol 0.1% group. Ophtalmologists should be aware that even PF formulations may lead to a mild ocular surface impairment.