Background Pre-operative MRI examination is currently considered as an evolving tool for assessment of perianal fistula types, extensions, and complications to achieve proper treatment plan. Proper assessment of fistula activity can also have a major contributing role to the treatment plan, through deciding the type of prescribed medications, proper surgical approach as well as the proper operative intervention timing. Reviewing literature, only few studies have mentioned the importance of diffusion tensor imaging (DTI) sequences in diagnosing perianal fistula activity, yet many studies have discussed the perianal fistula activity assessment using other diffusion weighted imaging (DWI) sequences. In the present study, the main objective was to prove the MRI quantitative DTI sequences’ ability of diagnosing perianal fistulae inflammatory activity. Results This study was a prospective analysis in which fistular activity was confirmed by intra-operative findings (considered as the standard reference). The cases included in the study were divided into two groups, based on their surgical findings, positive inflammatory and negative inflammatory groups. Both groups were pre-operatively assessed using MRI imaging and additionally used diffusion weighted and tensor imaging (DWI and DTI) sequences by special post-processing quantitative assessment of DTI FA and ADC values. There was significant statistical difference between the mean ADC value of the PIA and NIA groups in the track, edema, ipsilateral and contralateral sphincter areas with P values (P = 0.000, 0.000, 0.002 and 0.000 respectively). There was also significant difference between the mean FA value of the PIA and NIA groups in the track, edema, ipsilateral and contralateral sphincter areas with P values (P = 0.000, 0.000, 0.000 and 0.008 respectively). Conclusion This study results revealed that FA are relatively lower in positive inflammatory activity lesions than in negative inflammatory activity lesions while the ADC values were relatively higher in positive inflammatory activity lesions than in negative inflammatory activity lesions and the differences were statistically significant having a fundamental role in the assessment of perianal fistula activity especially at the track area being of highest sensitivity and specificity. Unlike conventional MRI sequences which revealed only high specificity being a good negative modality.