Introduction The use of bowel preparation strategies to reduce the degree of rectal distension during prostate radiotherapy is well documented. This retrospective pilot study analysed and compared a probiotic agent against a psyllium-supplemented diet to establish the feasibility of probiotics as a bowel preparation for patients receiving radical radiotherapy for prostate cancer. Method A retrospective chart review of five patients taking probiotics and five taking psyllium husk (psyllium) during their course of radiotherapy treatment was conducted. On treatment, cone beam computed tomography (CBCT) scans were compared with planning CTs to quantify inter-fractional variation in rectal volume and distension. Results Forty-five CBCT scans were available in both the psyllium and probiotics groups for analysis. Variation in mean difference in rectal volume from planning (?RV), mean rectal cross-section area (CSA) and mean relative cross-section area (CSArel) was significantly increased for the probiotics group compared with the psyllium group (p=0?001, 0?008 and 0?007, respectively). No statistically significant differences in mean ?RV, CSA and CSArel were detected between the two groups. Conclusion This retrospective analysis suggests that a probiotics-based bowel preparation that utilises Lactobacillus acidophilus and Bifidobacterium lactis may result in increased rectal volume and CSA variation throughout treatment in comparison with a psyllium-supplemented diet.