To evaluate the effects of the discontinuation of antithrombotic drugs on intraocular pressure (IOP) reduction and complications from ab interno trabeculotomy for patients with glaucoma. We performed a retrospective chart review on the data of patients treated with antithrombotic agents who have undergone ab interno trabeculotomy through Tanito microhook combined with cataract surgery at the Asahi General Hospital and the Tokyo University Hospital, with 6 months of follow-up. The patients were classified into two groups depending on whether they discontinued (AT-) or continued (AT+) antithrombotic therapy during the perioperative phase. The demographics, pre- and postoperative IOP, medication score, best-corrected visual acuity (BCVA), and postoperative complications were analyzed preoperatively and postoperatively at 1 week and 1–6 months. The series included 44 eyes from 44 Japanese patients. The AT- and AT+ groups included 21 eyes from 21 patients and 23 eyes from 23 patients, respectively. The decrease in IOP from the baseline at 1 week postoperative was significantly different between the two groups (p = 0.009), but there were no significant differences observed in the other visits. Hyphema and IOP spikes exceeding 30 mmHg occurred in 10% and 10% of AT- participants, and in 43% and 26% of AT+ participants, respectively. Hyphema and spikes with hyphema occurred more frequently in the AT+ than in the AT- group (p = 0.02 and p = 0.05). The number of patients who had spikes was not significantly different (p = 0.27). In trabeculotomy using the Tanito microhook®, discontinuing antithrombotic therapy had better IOP-lowering effects and less postoperative complications.