Preservative-free topical medications have been introduced for glaucoma care to reduce ocular adverse events associated with preservatives. This is a systematic review and meta-analysis of randomized clinical trials (RCTs) comparing the efficacy and safety of beta-blockers, or combination using beta-blockers, with and without preservatives. PubMed, EMBASE and Web of Science were examined. Risk of bias was assessed using the Cochrane Handbook for Systematic Reviews. The primary outcome was change in intraocular pressure (IOP) from baseline to final follow-up. Secondary outcomes included ocular and systemic side effects, and other clinical and quality of life outcomes. Of 242 records identified, seven RCTs (1125 patients) were included. The follow-up period ranged from one to 12 months. Timolol was used in five studies, and two studies used a combination (timolol with bimatoprost or dorzolamide). The difference in mean change (MD) in IOP between the preservative-free and the preserved drugs was statistically significant but not clinically relevant: (MD 0.29 mmHg, 95% confidence interval 0.07–0.51 mmHg, p = 0.010; moderate-certainty evidence). Regarding adverse events: Level of evidence for all ocular surface outcome was low or very low and reported in few studies. No significant difference was observed on ocular surface symptoms. Tear break-up time (TBUT) was better with preservative-free drops (p < 0.001). Schirmer’s test was better in the preservative-free group (p < 0.001). Level of evidence for all ocular surface outcomes was low or very low. There was no difference in other secondary outcomes. We found no clinically relevant difference in mean change in IOP between the preserved and the preservative-free treatments. Data on adverse events used different methods and were incompletely reported. Although some measures of ocular surface health favoured preservative-free medications, more evidence is needed. The increasing use of preservative-free drops may be associated with better ocular surface and tolerability, but strong evidence from RCTs would be welcome.