A series of 28 compounds, 3-nitro-1 -1,2,4-triazole, were synthesized by click-chemistry with diverse substitution patterns using medicinal chemistry approaches, such as bioisosterism, Craig-plot, and the Topliss set with excellent yields. Overall, the analogs demonstrated relevant in vitro antitrypanosomatid activity. Analog (R = 4-OCF -Ph, IC = 0.09 μM, SI = >555.5) exhibited an outstanding antichagasic activity ( , Tulahuen LacZ strain) 68-fold more active than benznidazole (BZN, IC = 6.15 μM, SI = >8.13) with relevant selectivity index, and suitable LipE = 5.31. was considered an appropriate substrate for the type I nitro reductases (TcNTR I), contributing to a likely potential mechanism of action for antichagasic activity. Finally, showed nonmutagenic potential against strains (TA98, TA100, and TA102). Therefore, 3-nitro-1 -1,2,4-triazole is a promising antitrypanosomatid candidate for in vivo studies.