Multidrug-resistant Tuberculosis (MDR-TB), caused by Mycobacterium tuberculosis strains resistant to at least two key anti-mycobacterial drugs, is an escalating global health concern. This necessitates the exploration of alternative therapeutic options. The Siddha system of medicine offers a variety of formulations with potential efficacy against severe infections such as TB. This study investigates the effectiveness of Serankottai nei, a Siddha medicinal formulation, against MDR-TB. Different concentrations of Serankottai nei (2%, 4%, and 6%) were evaluated in vitro for their anti-mycobacterial activity against an MDR-TB isolate (DKU-156), the reference strain M. tuberculosis H37Rv, and the rapid-growing mycobacterial pathogen M. fortuitum (TMC-1529) using Lowenstein-Jensen (L-J) medium. The activity was assessed by calculating the percentage inhibition based on the reduction in the number of colonies on drug-containing media compared to drug-free controls. Serankottai nei demonstrated significant anti-tuberculosis activity across all tested concentrations. For the MDR isolate DKU-156, the inhibition rates were 90%, 100%, and 100%, respectively. For M. tuberculosis H37Rv, inhibition rates were 86%, 98%, and 100%, while for M. fortuitum (TMC-1529), all concentrations achieved 100% inhibition in L-J medium. These results indicate that Serankottai nei exhibits promising anti-tubercular activity, warranting further ethno-pharmacological studies and drug development efforts for MDR-TB treatment.