Novel benzothiazole‒urea hybrids were designed, synthesized and evaluated their anti-bacterial activity. They only exhibited anti-bacterial activity against Gram-positive bacteria, including clinical methicillin-resistant S. aureus (MRSA), compounds 5f, 5i, 8e, 8k and 8l exhibited potent activity (MIC = 0.39 and 0.39/0.78 μM against SA and MRSA, respectively). Crystal violet assay showed that compounds 5f, 8e and 8l not only inhibited the formation of biofilms but also eradicated preformed biofilms. Compound 8l had membrane disruption, little propensity to induce resistance, benign safety and in vivo anti-MRSA efficacy in a mouse model of abdominal infection. Therefore, our data demonstrated the potential to advance benzothiazole‒urea hybrids as a new class of antibiotics.