The quest for novel antibacterial agents is imperative in the face of escalating antibiotic resistance. Naturally occurring tetrahydro-β-carboline (THβC) alkaloids have been highlighted due to their significant biological derivatives. However, these structures have been little explored for antibacterial drugs development. In this study, a series of 1,2,3,4-THβC derivatives were synthesized and assessed for their antibacterial prowess against both gram-positive and gram-negative bacteria. The compounds exhibited moderate to good antibacterial activity, with some compounds showing superior efficacy against gram-positive bacteria, especially methicillin-resistant Staphylococcus aureus (MRSA), to that of Gentamicin. Among these analogs, compound 3k emerged as a hit compound, demonstrating rapid bactericidal action and a significant post-antibacterial effect, with significant cytotoxicity towards human LO2 and HepG2 cells. In addition, compound 3k (10 mg/kg) showed comparable anti-MRSA efficacy to Ciprofloxacin (2 mg/kg) in a mouse model of abdominal infection. Overall, the present findings suggested that THβC derivatives based on the title compounds hold promising applications in the development of antibacterial drugs.