To assess clinical and biomolecular changes of the conjunctival epithelium in anophthalmic patients wearing an ocular prosthesis. Thirty-five unilateral anophthalmic patients were enrolled. Patients with blepharitis, lid abnormalities, and topical/systemic medication affecting the ocular surface were excluded. Symptom Assessment in Dry Eye (SANDE) questionnaire and tear function test (Schirmer Test Type I) were recorded. Conjunctival inflammation and meibomian gland dysfunction (MGD) were graded in the anophthalmic side and fellow eye. Impression cytology sampling of the upper, lower tarsal, and posterior/bulbar conjunctiva from the anophthalmic socket were collected and compared to healthy controls. Patients had significantly higher SANDE (p < 0.001), Schirmer I test (p = 0.004), conjunctival inflammation (p < 0.001), and MGD scores (p < 0.001) on the anophthalmic side compared to the fellow eye. Mucin 5AC, inflammatory markers (MMP-9, ICAM-1) expression (p < 0.001), and response to oxidative stress (NRF2-KEAP1 signaling pathway) (p < 0.05) were significantly upregulated in the posterior conjunctival surface in the anophthalmic socket. Anophthalmic patients complained of more pronounced dry eye symptoms and presented more significant signs of inflammation and MGD on the anophthalmic side. The bulbar conjunctiva, behind the prosthesis, showed more significant hyperexpression of mucins, markers of inflammation, and increased response to oxidative stress compared to the tarsal conjunctiva. Patients wearing ocular prosthesis had signs of inflammation resembling dry eye disease.