In this study, we report the design and synthesis of a series of novel thiophene-arylamide compounds derived from the noncovalent decaprenylphosphoryl-β-d-ribose 2'-epimerase (DprE1) inhibitor TCA1 through a structure-based scaffold hopping strategy. Systematic optimization of the two side chains flanking the thiophene core led to new lead compounds bearing a thiophene-arylamide scaffold with potent antimycobacterial activity and low cytotoxicity. Compounds <b>23j</b>, <b>24f</b>, <b>25a</b>, and <b>25b</b> exhibited potent <i>in vitro</i> activity against both drug-susceptible (minimum inhibitory concentration (MIC) = 0.02-0.12 μg/mL) and drug-resistant (MIC = 0.031-0.24 μg/mL) tuberculosis strains while retaining potent DprE1 inhibition (half maximal inhibitory concentration (IC<sub>50</sub>) = 0.2-0.9 μg/mL) and good intracellular antimycobacterial activity. In addition, these compounds showed good hepatocyte stability and low inhibition of the human ether-à-go-go related gene (hERG) channel. The representative compound <b>25a</b> with acceptable pharmacokinetic property demonstrated significant bactericidal activity in an acute mouse model of tuberculosis. Moreover, the molecular docking study of template compound <b>23j</b> provides new insight into the discovery of novel antitubercular agents targeting DprE1.