Purpose To evaluate the ocular surface, meibomian gland alterations, and in vivo confocal microscopy (IVCM) parameters through correlation with nicotine dependency level (NDL) and duration of smoking (DS) in chronic smokers. Methods This cross-sectional study included 50 eyes of 50 chronic smokers, and 50 eyes of 50 age- and gender-matched healthy nonsmokers. To examine the ocular surface and tear film, corneal sensitivity (CS) measurement with Cochet-Bonnet esthesiometer, tear film break-up time (TBUT), lissamine green (LG) staining, Schirmer I test with anesthesia, and Ocular Surface Disease Index (OSDI) questionnaire were performed consecutively. Basal epithelial cell, keratocyte, and endothelial cell density, the percentage of endothelial polymegethism/pleomorphism, and subbasal nerve plexus were evaluated using IVCM. Meibomian gland density was evaluated with a meibography unit. The Fagerstrom test was used to measure NDL, and DS was recorded. Results In the chronic smokers group, the basal epithelial cell density, anterior and posterior keratocytes, endothelial cell density, and long and total subbasal nerve numbers were lower, and LG staining, meiboscore, and OSDI scores were higher, compared with the control group, whereas other parameters were similar. The percentage of polymegethism was higher and the percentage of pleomorphism was lower in the chronic smokers group. In the correlation analysis, no significant relationship was found between the DS, NDL and ocular surface, and IVCM findings. Conclusion Decreased corneal basal epithelium, anterior and posterior keratocytes, endothelial cell density, meibomian gland density, and subbasal nerve numbers were found in chronic smokers. The results of the study show that smoking has an adverse effect on ocular surface parameters.