Tuberculosis remains one of the leading infectious diseases worldwide, which is why new approaches are being developed as a foundation for future diagnostic methods. One promising strategy relies on porphyrins as biomarkers for the detection of Mycobacterium tuberculosis using fluorescence spectroscopy. Previous attempts have been made to detect microorganisms based on their porphyrin production. However, insufficient endogenous porphyrin production limits the applicability of this approach for mycobacterial detection. As a proof-of-principle study, we investigated the feasibility of using exogenous precursors to enhance porphyrin accumulation with the aim of detecting mycobacterial growth by means of fluorescence spectroscopy. The introduction of these precursors resulted in a significantly higher fluorescence signal intensity, making it possible to detect Mycobacterium tuberculosis and identify potential contamination.