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Despite the efficient anti-cancer capabilities of methotrexate (MTX), it may induce myelosuppression, liver dysfunction and testicular toxicity. The purpose of this investigation was to determine whether Marrubium alysson L. (M. alysson L.) methanolic extract and its polyphenol fraction could protect mouse testicles from MTX-induced damage. We also investigated the protective effects of three selected pure flavonoid components of M. alysson L. extract. Mice were divided into seven groups (n = 8): (1) normal control, (2) MTX, (3) Methanolic extract + MTX, (4) Polyphenolic fraction + MTX, (5) Kaempferol + MTX, (6) Quercetin + MTX, and (7) Rutin + MTX. Pre-treatment of mice with the methanolic extract, the polyphenolic fraction of M. alysson L. and the selected pure compounds ameliorated the testicular histopathological damage and induced a significant increase in the serum testosterone level and testicular antioxidant enzymes along with a remarkable decline in the malondialdehyde (MDA) level versus MTX alone. Significant down-regulation of nuclear factor kappa B (NF-κB), tumor necrosis factor-alpha (TNF-α), p53 and miRNA-29a testicular expression was also observed in all the protected groups. Notably, the polyphenolic fraction of M. alysson L. displayed a more pronounced decline in the testicular levels of interleukin-1β (IL-1β), interleukin-6 (IL-6) and MDA, with higher testosterone levels relative to the methanolic extract. Further improvements in the Johnsen score, histopathological results and all biochemical assays were achieved by pre-treatment with the three selected pure compounds kaempferol, quercetin and rutin. In conclusion, M. alysson L. could protect against MTX-induced testicular injury by its antioxidant, anti-inflammatory, antiapoptotic activities and through the regulation of the miRNA-29a testicular expression. The present study also included chemical profiling of M. alysson L. extract, which was accomplished by LC-ESI-TOF-MS/MS analysis. Forty compounds were provisionally assigned, comprising twenty compounds discovered in the positive mode and seventeen detected in the negative mode.