Background The length of sphincter which can be divided during fistulotomy for perianal fistula is unclear. The aim was to quantify sphincter damage during fistulotomy and determine the relationship between such damage with symptoms and severity of faecal incontinence and long-term quality of life (QOL). Methods A prospective cohort study was performed over a 2-year period. Patients with intersphincteric and mid to low transsphincteric perianal fistulas without risk factors for faecal incontinence were scheduled for fistulotomy. All patients underwent 3D endoanal ultrasound (3D-EAUS) pre-operatively and 8 weeks postoperatively. Measurements were taken of pre- and postoperative anal sphincter involvement and division. Anal continence was assessed using the Jorge-Wexner scale and QOL scores pre, 6 and 12 months postoperatively. Results Forty-nine patients were selected. A strong correlation between pre- and postoperative measurements was found p < 0.001. A median length of 41% of the external anal sphincter and 32% of the internal anal sphincter was divided during fistulotomy. Significant differences in mild symptoms of anal continence were found with increasing length of external anal sphincter division. But there was no significant deterioration in continence, soiling, or quality of life scores at the 1-year follow-up. Division of over two-thirds of the external anal sphincter was associated with the highest incontinence rates. Conclusions 3D-EAUS is a valuable tool for quantifying the extent of sphincter involvement pre- and postoperatively. Post-fistulotomy faecal incontinence is mild and increases with increasing length of sphincter division but does not affect long-term quality of life.