A series of pyrido[2,3-d][1,2,4]triazolo[4,3-a]pyrimidin-5-ones (8) has been synthesised via reaction of 5-substituted-2-thioxo-2,3-dihydro-1H-pyrido[2,3-d]pyrimidin-4-one (3) or its methylthio derivative 4 with hydrazonoyl chlorides 5. Alternative syntheses of products 8 were carried out either by reaction of enaminone 1 with 7-amino-1,3-disubstituted[1,2,4]triazolo[4,3-a]pyrimidin-5-one (10) or via the Japp-Klingemann reaction of compound 13. Both conventional thermal and microwave irradiation techniques were used for synthesis of the target products 8 and a comparative study of these techniques using triethylamine or chitosan, as basic catalysts, was carried out. The mechanisms of the reactions under investigation are discussed. In addition, the antimicrobial activity of the newly synthesized products was evaluated.