Today, the emergence of antibiotic resistance in pathogenic bacteria is considered an important problem for society. Excessive consumption of antibiotics, long-term treatments, and inappropriate prescriptions continually increase the severity of the problem. Improving antibiotic stewardship requires improved diagnostic testing, and, therefore,in vitroantibiotic susceptibility testing is becoming increasingly important. This research details the development of an antibiotic susceptibility test forMycobacterium smegmatisusing streptomycin as antibiotics. This strain was selected because it is a member of the slow growingMycobacteriumgenus and serves as a useful surrogate organism forM. tuberculosis. A commercially available and low-cost screen-printed gold electrode in combination with a specifically developed nucleic acid probe sequence for the 16SrRNA region of the mycobacterial genome was employed to monitorM. smegmatisnucleic acid sequences using the techniques of square-wave voltammetry and electrochemical impedance spectroscopy. The results show that it was possible to detectM. smegmatissequences and distinguish antibiotic-treated cells from untreated cells with a label-free molecular detection. As a result, thein vitroantibiotic susceptibility test revealed thatM. smegmatisshowed sensitivity to streptomycin after a 24-H incubation, with the developed protocol representing a potential approach to determining antibiotic susceptibility more quickly and economically than current methods.