Introduction: Anti-glaucoma medication is the first line treatment modality in Primary Open Angle Glaucoma (POAG) management, and evidence has shown that topically administered medications over a long time can cause structural changes on the ocular surface. This study aimed to determine the effect of 0.5% Timolol Maleate with preservative on Tear film of newly diagnosed glaucoma patients in Owerri, Imo State, Nigeria, with a view to making recommendation for optimal use of Timolol Maleate in the management of POAG patients. Methods: This was a hospital based prospective study which compared the Tear film break up time (TBUT) and tear production of newly diagnosed glaucoma patients on topical Timolol with those of normal healthy subjects (age and sex matched controls) who were not on any topical medications. Researcher administered questionnaire with subfields on respondent’s sociodemographics, patient’s history; measured TBUT and tear production was used. Descriptive and analytical statistics were performed. A p value of <0.05 was considered statistically significant. Results: A total of 140 eyes were examined. The mean TBUT and Schirmer’s test value showed statistically significant reduction from 1st month till 3rd month compared with baseline values. A significant proportion of Timolol group had unstable tear film, and this was statistically significant. Majority of the participants (70%) on Timolol had ocular discomfort while using the medication. Majority of them also had also peppery sensation (57.1%), followed by stinging sensation (38.8%) and then burning and foreign body sensation (30.6% each). There was no statistically significant difference between TBUT and Schirmer’s test value of participants with complaints and those without complaints. Conclusion and Recommendations: Abnormal TBUT and Schirmer’s test values were recorded amongst participants on Timolol and these values showed statistically significant reduction over the 3 months reviewed, suggesting that Timolol with preservatives affects TBUT and tear production. In addition, these patients also reported symptoms suggestive of ocular surface disease implying that Timolol with preservatives may be a risk factor for developing ocular surface disease and dry eyes. Assessment of patients for dry eyes prior to recommending Timolol with preservatives may be necessary to ensure compliance and optimal use of Timolol.