Trichomoniasis is the most prevalent, non-viral sexually transmitted diseases (STD) caused by amitochondriate protozoan Trichomonas vaginalis. Increased resistance of T. vaginalis to the marketed drug Metronidazole necessitates the development of newer chemical entities. A library of sixty 2-methyl-4/5-nitroimidazole derivatives was synthesized via nucleophilic ring opening reaction of epoxide and the efficacies against drug-susceptible and -resistant Trichomonas vaginalis were evaluated. All the molecules except two were found to be active against both susceptible and resistant strains with MICs ranging 8.55-336.70 μM and 28.80-1445.08 μM, respectively. Most of the compounds were remarkably more effective than the standard Metronidazole. This study analyzes the in vitro and in vivo activities of the new 5-nitroimidazoles, which were found to be safe against human cervical HeLa cells with good selectivity index. The exploration of SAR by the synthesis of four different prototypes and 3D-QSAR study has shown the importance of prototype 1 over other prototypes.