Abstract: Introduction: The quinazolin-4(3H)-one core is a crucial structural motif in medicinal chemistry, widely recognized for its exceptional chemical stability and a broad spectrum of pharmacological properties. Incorporating bioactive moieties via chemical modification, such as Mannich base synthesis, represents a powerful strategy for developing novel therapeutic agents.  Methods: In this study, a series of six novel quinazolin-4(3H)-one-based N-Mannich base derivatives (3A–3F) were designed and successfully synthesized through a green, microwave-assisted one-pot method by reacting quinazoline-4(3H)-one, formaldehyde, and various primary aromatic amines. The chemical structures of all synthesized compounds were validated using Fourier-transform infrared (FT-IR), nuclear magnetic resonance (1H-NMR), and high-resolution mass spectrometry (HRMS) analyses. The derivatives were evaluated in vitro for their anti-glioblastoma potential against U-87 Malignant Glioma cells using the MTT colorimetric assay, and for their anti-inflammatory activity using the egg albumin protein denaturation method with diclofenac sodium as a standard.  Results: The microwave-assisted synthesis delivered high yields ranging from 75% to 95%. In biological evaluations, compound 3E—selected via prior computational docking investigations demonstrated moderate concentration-dependent anti-glioblastoma activity with an IC50 value of 41.52 μg/mL. Furthermore, all six derivatives exhibited concentration-dependent anti-inflammatory efficacy by protecting protein from heat-induced denaturation. At the highest tested concentration (400 μg/mL), compound 3F exhibited the highest anti-inflammatory protection (87.58%), followed by 3E (83.00%) and 3D (80.71%), performing closely to the standard drug diclofenac (93.13%). Notably, at lower concentrations (100 μg/mL and 200 μg/mL), compounds 3E and 3F showed inhibition comparable to or higher than diclofenac at 100 μg/mL, and 3E matched diclofenac at 200 μg/mL.  Conclusion: The findings indicate that the nature of the aromatic amine substituents attached to the quinazolin-4(3H)-one core heavily modulates biological potency. These derivatives, particularly 3E and 3F, represent promising lead candidates for advanced multi-target drug development in oncology and inflammatory disorders.