The cholinergic system regulates a variety of structures on the ocular surface and maintains homeostasis. Therefore, dysregulation of cholinergic activity could impact lacrimal and conjunctival function. Consequently, exposure of the ocular surface to chemical agents, including pesticides such as acetylcholinesterase inhibitors, which interact closely with the ocular surface and anterior segment, could affect ocular homeostasis. In this study, we assess alterations in the ocular surface in workers exposed to organophosphate pesticides. A cross-sectional study was conducted involving 39 participants (23 controls and 16 cases exposed to agrochemicals). The ocular surface of all subjects was assessed using the Schirmer test, TBUT, conjunctival impression cytology, and the Q16 neurotoxicity and ocular symptoms questionnaire by Donate. Conjunctival hyperemia, impression cytology and Schirmer test were found to be altered in the workers exposed to cholinesterase inhibitor pesticides. These findings suggest that ocular surface alterations can be seen in organophosphate pesticide exposure, and that lacrimal hypersecretion could be an ocular biomarker for detecting pesticide effects before adverse clinical effects occur.