Aim Nitric oxide donors, such as isosorbide dinitrate ointment (ISDN), are considered as first-choice agents in the treatment of chronic anal fissure. Injection with botulinum toxin A in the internal anal sphincter is often used as a second-line therapy, although it may give better results and fewer side effects than nitric oxide donors. The aim of this randomized clinical trial was to investigate whether botulinum toxin A (Dysport®) is more effective than ISDN in the primary treatment of chronic anal fissure. Method From April 2005 until October 2009, 60 patients (32 men) with a median age of 42 (25–82) years were randomized to receive either ISDN 10 mg/ml (1%) (n = 33) or injection with 60 units of Dysport® (n = 27). The primary end-point was the percentage of complete fissure healing after 8 weeks. Results After a median of 9 weeks complete fissure healing was noted in 18 of 27 patients in the Dysport® group and in 11 of 33 patients in the ISDN group (P = 0.010). Absolute improvement of pain scores after 9 weeks was similar in both groups (P = 0.733). Patients treated with Dysport® had fewer side effects than patients treated with ISDN (P = 0.028). Of the patients with a healed fissure, 28% of the Dysport® group and 50% of the ISDN group had a recurrence within 1 year (P = 0.286; hazard ratio 2.08; 95% CI = 0.54–7.97). Conclusion Dysport® is more effective than ISDN ointment and has fewer side effects in the primary treatment of chronic anal fissure. The recurrence rate within 1 year in both treatment groups is high.