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PURPOSE To investigate the effects of mitomycin-C (MMC) and 5-fluorouracil (5-FU) on the viability, proliferation, and migratory capacity of cultured ocular adnexal sebaceous carcinoma (SC) cells. DESIGN Laboratory investigation. METHODS Human SC cell lines (Bascom Palmer 50 and 52 [BP50 and BP52]) and human limbal stem cells (LSCs) were treated with various concentrations of MMC and 5-FU. Cytotoxicity was assessed with the tetrazolium MTT colorimetric viability assay on normal corneal versus tumor cells. Growth curves and scratch assays were performed to characterize the effects of these chemotherapeutic agents on SC proliferation and migration, respectively. RESULTS MMC decreased BP52 cell viability in a dose-dependent manner with a half-maximal effective dose (EC50) of 11.8 μM after 72 hours. SC viability decreased >50% at 80 mM 5-FU after 72 hours. MMC reduced LSC viability in a dose-dependent manner with an EC50 value of 3.24 μM, and 5-FU decreased LSC viability >50% at 160 μM. MMC decreased SC cell proliferation and migration in a dose-dependent manner. 5-FU displayed antiproliferative effects but did not affect cell migration at concentrations below 1000 μM. CONCLUSIONS Our in vitro data corroborate clinical observations that MMC is efficacious for treating ocular adnexal SC, albeit at the expense of LSC viability. Our findings also demonstrate that topical 5-FU exhibits antiproliferative effects that supersede its cancer-killing and anti-migratory effects on cultured SC cells.