In the present investigation, a series of new Mannich bases were prepared by the reaction of 2-ethoxybenzaldehyde with isoniazid to form acid hydrazone (3a). Further, C-Mannich bases of the above acid hydrazone were prepared by aminomethylation with formaldehyde and substituted secondary amines (3b–3k). The structures of newly synthesized compounds were evaluated by elemental analyses and spectral (IR, 1H NMR, 13C NMR) studies. All the synthesized compounds were evaluated for their antimicrobial activity. Amoxicillin was used as a standard drug for antibacterial activity while Nystatin was used as a standard drug for antifungal activity. Preliminary pharmacological evaluation revealed that the compound (3f, 3i, 3j, 3k) showed better performance against Bacillus subtilis, Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Candida albicans and Candida gabrata. The result demonstrates the potential and importance of developing new mannich bases which would be effective against resistant bacterial and fungal strain.