Abstract Paclitaxel is a well-known chemotherapeutic agent that induces cancer cell death by stabilising microtubules. Avanafil, a phosphodiesterase type 5 (PDE5) inhibitor typically used for erectile dysfunction, has recently been proposed to enhance blood-brain barrier permeability. This research focuses on, for the first time, the role of avanafil used together with paclitaxel to treat glioma. The cytotoxic effect of this combination on the U373 brain cancer cell was evaluated using MTT, colony survival analysis, and wound healing assay. Western blot analyses of the agents were conducted to investigate their effects on p53, Cas-3, Cas-9, Bax, Bcl-2, and c-PARP proteins. Additionally, Avanafil, Paclitaxel, and Avanafil+Paclitaxel combinations were calculated using the Gaussian package program at the B3LYP, HF, and M062x levels in the 6–31 g, 6–31++g, and 6–31++g(d, p) basis sets. The activities of the agents against brain tumour proteins (PDB ID: 2DME and 6YPE) were compared and their ADME/T properties were also investigated. MTT and colony survival analysis demonstrated that the combination significantly inhibited U373 cell viability and proliferation. Wound healing and Western blot assays indicated impaired migration and altered expression of apoptosis-related proteins. Molecular docking and DFT analyses supported the experimental findings, and ADME/T profiling suggested favourable pharmacokinetics. Our results indicate that avanafil enhances the cytotoxicity of paclitaxel via non-apoptotic mechanisms, highlighting its potential as an adjuvant in glioma therapy.