Brain and other nervous system cancers are the 10th leading cause of death worldwide. Genome instability, cell cycle deregulation, epigenetic mechanisms, cytoarchitecture disassembly, redox homeostasis as well as apoptosis are involved in carcinogenesis. A diet rich in fruits and vegetables is inversely related with the risk of developing cancer. Several studies report that cruciferous vegetables exhibited antiproliferative effects due to the multi-pharmacological functions of their secondary metabolites such as isothiocyanate sulforaphane deriving from the enzymatic hydrolysis of glucosinolates. We treated human astrocytoma 1321N1 cells for 24 h with different concentrations (0.5, 1.25 and 2.5% v/v) of sulforaphane plus active myrosinase (Rapha Myr®) aqueous extract (10 mg/mL). Cell viability, DNA fragmentation, PARP-1 and γH2AX expression were examined to evaluate genotoxic effects of the treatment. Cell cycle progression, p53 and p21 expression, apoptosis, cytoskeleton morphology and cell migration were also investigated. In addition, global DNA methylation, DNMT1 mRNA levels and nuclear/mitochondrial sirtuins were studied as epigenetic biomarkers. Rapha Myr® exhibited low antioxidant capability and exerted antiproliferative and genotoxic effects on 1321N1 cells by blocking the cell cycle, disarranging cytoskeleton structure and focal adhesions, decreasing the integrin α5 expression, renewing anoikis and modulating some important epigenetic pathways independently of the cellular p53 status. In addition, Rapha Myr® suppresses the expression of the oncogenic p53 mutant protein. These findings promote Rapha Myr® as a promising chemotherapeutic agent for integrated cancer therapy of human astrocytoma.