In an effort to find new leads as anticancer or antimicrobial agents, the present work deals with the synthesis of some novel 1-substituted 1,2,4-triazolo[4,3-a]quinoxalines 7, 9a,b, and 14–19 and 1,2,4-triazino[4,3-a] quinoxalines 10a–c as well as 2-[5-amino-3-(4-chlorophenyl)pyrazol-1-yl]-3-benzylquinoxaline 13. These were synthesized using the key intermediate 3-benzyl-2-hydrazinoquinoxaline 6 with various reagents. Ten compounds, namely 7, 9a, 10b, 11, and 13–18 were chosen by the National Cancer Institute of Bethesda (NCI) for evaluation of their anticancer activity. The results indicated that 9a was the most active and was further evaluated for in vitro five dose assay against 60 human cell lines. It was proven to possess the highest broad spectrum anticancer activity. It showed particular effectiveness towards leukemia SR, non-small cell lung cancer HOP-92, NCI-H460, colon cancer HCT-116, HCT-15, CNS cancer U251, melanoma LOX IMVI, renal cancer A498, prostate cancer PC-3, and breast cancer MDA-MB-468 cell lines (GI50 = 3.91, 3.45, 3.49, 3.21, 1.96, 5.18, 3.69, 1.80, 5.19, and 5.55 mM, respectively). All new compounds were screened for their antimicrobial activity and were very active against P. aeruginosa. Compounds 10a and 16 were twice as active as ampicillin against P. aeruginosa. Five compounds, 9a, b, 10b, 13, and 14 were equipotent to ampicillin against P. aeruginosa. In addition, compound 16 showed a broad spectrum antimicrobial activity. Furthermore, compound 9a showed dual activity as an anticancer and antimicrobial agent.