A series of pyrazinamide (PAZ) Mannich bases has been synthesized by reacting PAZ, formaldehyde, and various substituted piperazines using microwave irradiation with the yield ranging from 46% to 86%. The synthesized compounds were evaluated for antimycobacterial activity in vitro and in vivo against Mycobacterium tuberculosis H37Rv (MTB). Among the synthesized compounds, 1-cyclopropyl-6-fluoro-1,4-dihydro-8-methoxy-7-(3-methyl-4-((pyrazine-2-carboxamido)methyl)piperazin-1-yl)-4-oxoquinoline-3-carboxylic acid (17) was found to be the most active compound in vitro with MIC of 0.39 and 0.2 microg/mL against MTB and multidrug-resistant MTB, respectively. In the in vivo animal model 17 decreased the bacterial load in lung and spleen tissues with 1.86 and 1.66-log10 protections, respectively.