Chronic anal fissure (CAF) is perpetuated by high sphincter pressures and secondary local ischemia. Pharmacological approaches include topical nitrates and botulinum toxin (BT), which both help to decrease the sphincter pressure. The aims of the present study were to assess the efficacy and safety of BT injection and combined treatment with BT injection and lowered dose glyceryltrinitrate (GTN) cream for the treatment of CAF. We hypothesised that combined treatment would have a synergistic effect on healing. Forty-one consecutive patients with CAF were randomly assigned to receive one of the following treatments: Group A, injection of BT (20 U into internal anal sphincter) and Group B, BT injection (20 units) and subsequent thrice daily topical applications of half-dose 0.2% GTN cream for 6 weeks. Patients were followed up at 6 and 12 weeks and were assessed for healing of anal fissure, by means of visual inspection using fissure grades; for faecal incontinence, using Cleveland Clinic incontinence scores; and for fissure pain and headache using a numeric pain rating scale. Fissure healing was similar in the two groups at both 6 (30% in BT and GTN and 33% in BT only) and 12 weeks (50% in BT and GTN vs 57% in BT-only group). Neither the change in pain score from 6 to 12 weeks, nor the overall level of pain was significantly different in the 2 groups. Moderate or severe headaches were suffered by 58% of patients using GTN. Single-agent treatment by means of BT injection alone was well tolerated compared with combination treatment with BT injection and GTN cream, with no significant differences in healing of CAF observed in this small study.