Background: The aim of the current study is to evaluate the safety, tolerability, and efficacy of neoadjuvant MMC in patients (pts) with non-muscle invasive bladder cancer (NMIBC). Methods: This is a prospective phase III randomized clinical trial in pts with primary clinical diagnosis of urinary bladder cancer or secondary recurrent untreated bladder cancer since May 2022 to September 2023 (EudraCT 2021-003751-42_studio ICH-013-). Pts are randomized 1:1 to neoadjuvant MMC arm (neoA) or standard arm (StA). In the 2 weeks before scheduled TUR (day 0) neoA pts receive intravesical instillations of MMC (40 mg/40 ml saline) at day -14 and -7 and cystoscopy with a cold cup biopsy at -14. After TUR, clinical choices both in the two groups, depend on histological evaluation of the tumour and EAU guidelines. Midstream and catheter urines are collected before instillations, at day 0 and after 3 months to respectively measure the level of specific biomarker (i.e., HMGB1) and to identify the microbiota. The expression of a mitomarker is evaluated on tissue samples. The primary endpoint is to assess safety, tolerability, and efficacy of neoadjuvant MMC in reducing the recurrence rate of BC calculated as the proportion of pts who achieve a complete response (no evidence of BC after 3, 6, 12 and 24 mo). The secondary endpoint will be the analysis of the rate of grade and stage progression to MIBC in case of recurrence and the correlation with biomarkers. Results: The interim analysis (median follow-up 6 months) included 54 pts: 26 in the StA and 28 in the neoA. Respectively in StA and neoA mean age at TUR was 66.5 (±9.9) and 65.8 (±9.9), males were 23 (88.46%) and 24 (85.71%). No statistically significant difference was observed between groups. 5 neoA pts [17.86% (95%CI 6.06-36.89)] after instillations reported 4 different adverse reactions, grade 1 of Clavien Dindo classification. NeoA pts showed higher level of HMGB1 at day 0 and after 3 months, suggesting an increased induction of immunogenic cell death (ICD). Shotgun metagenomics on microbial DNA from catheter urine is still ongoing. In field recurrence has been observed in 1 (3.85%) StA and 2 (7.14%) neoA pts. Conclusions: Preliminary data show neoadjuvant MMC to be safe and well tolerated and seem to correlate with increase of HMGB1. The short follow-up and the small sample size do not allow conclusion on the efficacy. Clinical trial information: EUCTR2021-003751-42.