Background: Perianal Crohn’s fistula and their response to anti-tumour necrosis factor (TNF) therapies are best assessed with magnetic resonance imaging (MRI), but radiologist reporting is subjective and variable. This study investigates whether segmentation software could provide precise and reproducible objective measurements of fistula volume. Methods: Retrospective analysis of patients with perianal Crohn’s fistula at our institution between 2007 and 2013. Pre- and post-biologic MRI scans were used with varying time intervals. A total of two radiologists recorded fistula volumes, mean signal intensity and time taken to measure fistula volumes using validated Open Source segmentation software. A total of three radiologists assessed fistula response to treatment (improved, worse or unchanged) by comparing MRI scans. Results: A total of 18 cases were reviewed for this pilot study. Inter-observer variability was very good for volume and mean signal intensity; intra-class correlation (ICC) 0.95 [95% confidence interval (CI) 0.91–0.98] and 0.95 (95% CI 0.90–0.97) respectively. Intra-observer variability was very good for volume and mean signal intensity; ICC 0.99 (95% CI 0.97–0.99) and 0.98 (95% CI 0.95–0.99) respectively. Average time taken to measure fistula volume was 202 s and 250 s for readers 1 and 2. Agreement between three specialist radiologists was good [kappa 0.69 (95% CI 0.49–0.90)] for the subjective assessment of fistula response. Significant association was found between objective percentage volume change and subjective consensus agreement of response ( p = 0.001). Median volume change for improved, stable or worsening fistula response was −67% [interquartile range (IQR): −78, −47], 0% (IQR: −16, +17), and +487% (IQR: +217, +559) respectively. Conclusion: Quantification of fistula volumes and signal intensities is feasible and reliable, providing an objective measure of perianal Crohn’s fistula and response to treatment.