Background <p>Isotretinoin is a vitamin A derivative used as a first-line treatment for moderate to severe acne. One of its most frequent side effects is dry eye disease (DED), in which tear film instability and neurosensory abnormalities play an etiologic role. The purpose of this research was to analyze changes in corneal innervation and its association with tear film integrity in patients treated with oral isotretinoin.</p> Methods <p>Twenty-eight patients treated with oral isotretinoin and the same number of age and sex distribution-matched healthy volunteers were included in this observational case‒control study. They were examined by in vivo corneal confocal microscopy (IVCM) to quantify the corneal nerve fiber density (CNFD), corneal nerve branch density (CNBD), corneal nerve fiber length (CNFL), and corneal nerve fractal dimension (CNFrD) in addition to the presence of dendritiform cells, microneuromas, and visibility of axonal beadings. Tear film analysis was also performed, as was the perception of discomfort related to ocular surface disease.</p> Results <p>The results of the comparative analysis of the isotretinoin group vs. the control group (mean [standard error]) were as follows: CNFD 18.57 [1.67] vs. 24. 57 [1.55] fibers per mm² (<i>p</i> = 0.01); CNBD 19.05 [2.43] vs. 30.62 [3.53] branches per mm² (<i>p</i> = 0.02); CNFL 13.90 [1.01] vs. 14.64 [0.66] mm per mm² (<i>p</i> = 0.54); CNFrD 1.46 [0.005] vs. 1.49 [0.006] (<i>p</i> = 0.01); Schirmer test 5.7 [1.1] vs. 10.3 [0.5] millimeters (<i>p</i> &lt; 0.001). The break-up time (BUT) was 5.70 [0.6] vs. 11.2 [0.2] seconds (<i>p</i> &lt; 0.001). A linear regression equation model was developed for BUT (R² = 0.48; 1-β = 0.98; <i>p</i> = 0.01) and CNFrD (R² = 0.58; 1-β = 0.99; <i>p</i> = 0.002).</p> Conclusion <p>Oral isotretinoin treatment promotes a vicious cycle of ocular surface disease that leads to damage to the subbasal nerve plexus. This damage increases with treatment time and impacts basal tear production.</p>