In the present work, the synthesis and biological activities of substituted 2-aminopyridine δ-lactone derivatives were achieved. 4,6,6-trimethyl-2-oxo-5,6-dihydro-2H-pyran-3-carbonitrile was synthesised from 4-hydroxy-4-methylpentan-2-one, followed by its transformation in enaminonitrile with DMFDMA. Then, the latter can react with various amines giving substituted 2-aminopyridine δ-lactones characterised by spectroscopic methods (IR, ¹H NMR, ¹³C NMR and MS). The antioxidant effects of substituted 2-aminopyridine δ-lactone derivatives were evaluated through DPPH assay and revealed a great antioxidant capacity. The antifungal and antibacterial activities were investigated by disc diffusion method against clinical Gram-negative bacteria and against clinical fungi. The study shows moderate to very good antibacterial and antifungal activities for the new substituted 2-aminopyridine δ-lactone derivatives.