A series of novel 1,2-naphthoquinones possessing tetrazolo[1,5-a]pyrimidine scaffolds were synthesized in very good yields using one-pot condensation of 2-hydroxy-1,4-naphthoquinone, aldehydes, and 5-aminotetrazole. All the synthesized compounds were evaluated for their antitumor activity in vitro against HCT116 (human colon cancer) and HepG2 (human hepatoma) cell lines. Among the screened compounds, 4o, 4k, 4g, 4q, and 4s showed significant inhibitory activity against the two human cell lines.