A Streptomyces strain MS-6-6 with promising anti-tuberculous activity was isolated from soil samples in Saudi Arabia. The nucleotide sequence of its 16S rRNA gene (1426 bp) evidenced a 100% similarity to Streptomyces mutabilis. Through an anti-tuberculous activity-guided approach, a polyketide macrolide was isolated and identified as treponemycin (TP). The structure of the isolated compound was determined by comprehensive analyses of its 1D and 2D NMR as well as HRESI-MS. In addition to the promising anti-tuberculous activity (MIC = 13.3 µg/mL), TP showed broad spectrum of activity against the Gram positive, Gram negative strains, and Candida albicans. Improvement of TP productivity (150%) was achieved through modification in liquid starch nitrate medium by replacing KNO3 with corn steep liquor and yeast extract or tryptone, and removing CaCO3 and K2HPO4. The follow up of TP percentage as well as its metabolites profile for each media was assessed by LC/DAD/MS.