In this study, a new series of barbituric acid-derived hybrid compounds (S1–S10) was synthesized through a multi-step synthetic protocol. The synthesis began with a Knoevenagel condensation reaction between barbituric acid and substituted aromatic aldehydes (4-hydroxybenzaldehyde or 4-chlorobenzaldehyde) in the presence of anhydrous sodium acetate to produce compounds S1 and S2. This was followed by Michael addition with acrylonitrile in the presence of triethylamine to obtain compounds S3 and S4. Cyclization with semicarbazide under acidic conditions led to the formation of 1,3,4-oxadiazole derivatives S5 and S6, followed by Schiff base formation with 4-(dimethylamino) benzaldehyde to produce compounds S7 and S8. Finally, a [3+2] cycloaddition reaction with sodium azide afforded the final compounds S9 and S10 containing dihydrotetrazole moieties. The structures of all synthesized compounds were confirmed by FT-IR, 1H-NMR, and 13C-NMR spectroscopic techniques. Compounds S9 and S10 were evaluated for their antibacterial activity against Staphylococcus aureus and Escherichia coli, antifungal activity against Candida albicans, antioxidant activity using the DPPH free radical scavenging assay, and anticancer activity against CaCo-2 cells. The obtained results demonstrated that compounds S9 and S10 possessed promising biological activities, suggesting a significant relationship between chemical structure and biological activity. Furthermore, POM and in silico ADME studies were performed to predict toxicity risks, physicochemical properties, pharmacokinetic behavior, and drug-likeness profiles of the synthesized compounds. These findings suggest that the synthesized hybrid derivatives may serve as promising candidates for further development as antimicrobial and anticancer agents.