Autologous serum eyedrops (ASE) are effective in treating various ocular surface diseases, including damages induced by long-term use of preserved glaucoma eyedrops. However, there has been no study on whether ASE is effective without stopping the causative eyedrops. This retrospective observational study included 55 patients with ocular-surface diseases caused by long-term use of preserved glaucoma eyedrops: 18 patients who used ASEs for 2 months without discontinuing the use of glaucoma eyedrops (Group 1), 22 patients who used ASEs for 2 months, discontinuing the use of glaucoma eyedrops for the first month (Group 2) and 15 patients who used non-preservative artificial tears for 2 months, discontinuing the use of glaucoma eyedrops for the first month (Group 3). There were no intergroup differences in the baseline values of the Schirmer I test results, tear breakup time (TBUT), ocular surface staining (OSS) score, loss of the meibomian gland, meibum quality and ocular-surface disease index (OSDI). Group 1 showed significant differences in TBUT, OSS score and OSDI at 2 months when compared to the baseline values before treatment, while Group 2 showed significant differences in those values at both 1 and 2 months. There were no differences in any of the parameters at baseline, 1 month or 2 months in Group 3. Our result suggested that ASE is effective for treating ocular surface diseases caused by glaucoma eyedrops containing preservatives and its effects can be expected without interruption of glaucoma eyedrop treatment.