Abstract Glioblastoma (GBM) remains the most lethal brain tumor with limited treatment options and poor prognosis. The design of new anti-GBM drug candidates is of utmost need. Here we assessed the activity of 1,2,4-triazolo[3,4-a]phthalazine and 1,2,4-triazolo[4,3-b]pyridazine derivatives in GBM cell lines, patient-derived primary GBM cells as well as normal human astrocytes (NHA). The tested compounds presented considerable cytotoxicity against GBM cells in MTT assay, and two of them were much less harmful to NHA. They also significantly decreased the ability of GBM cells to form colonies. The initial list of possible molecular targets was retrieved from the SuperPred tool and intersected with GBM-related genes extracted from publicly available datasets. The obtained records were subjected to further functional pathway enrichment analysis which identified histone modifying pathways among the most enriched ones. Network analysis revealed several putative molecular targets, including topoisomerase IIα (TOP2A). Molecular docking studies demonstrated relevant molecular interactions between the tested compounds and two macromolecules, TOP2A and bromodomain of the protein highly homologous to human PCAF, a member of the histone acetyltransferase family. These findings were supported by molecular dynamics simulation. ADME results indicated satisfactory pharmacokinetic parameters and revealed high drug-likeliness. Our results support further experimental validation of the studied compounds and their modifications which improve anti-GBM efficacy and selectivity.