Mycobacterium tuberculosis (M. tb), the causative agent of the infectious lung disease tuberculosis (TB), is estimated to infect approximately 1.7 billion people worldwide. This pathogen was responsible for more than 1.5 million deaths in 2020, and is likely to remain a global threat for many years to come due to the rising incidence of antibiotic resistance, as well as dramatic setbacks in treatment due to the ongoing COVID-19 pandemic. There is an urgent demand for novel therapeutics to treat the disease through unique mechanisms of action. In the search for these drugs, a novel collection of 101 marine actinomycetes previously isolated from a Caribbean giant barrel sponge Xestospongia muta was investigated for their ability to inhibit M. tb growth. Thirteen novel strains of Micrococcus, Micromonospora, Brevibacterium, and Streptomyces were identified as consistently producing extracts that inhibit M. tb in a dose-dependent manner. After sequencing the genomes of these strains, a comparative analysis between three assembly algorithms (SPAdes, A5-miseq, Shovill) was performed to determine which program yielded the best assembly from Illumina MiSeq data for biosynthetic gene cluster (BGC) mining. Upon characterizing the biosynthetic potential of each strain, two isolates generating highly potent extracts – Micrococcus sp. strain R8502A1 and Micromonospora sp. strain R45601 – were selected for further analysis through a dual genomics and chemistry-enabled approach. No compounds with obvious anti-TB activity were detected in the genome of Micrococcus sp. strain R8502A1, suggesting production of an elusive and novel anti-TB compound through a cryptic pathway. A comprehensive examination of all BGC-associated domains was conducted to evaluate possible biosynthetic pathways linked to the anti-TB activity observed. The active component of the Micrococcus extract was further isolated with high performance liquid chromatography (HPLC) and is under investigation with liquid chromatography-mass spectrometry (LC-MS) and nuclear magnetic resonance (NMR). In contrast, a BGC with 94% similarity to the selective and potent but poorly soluble anti-TB compound diazaquinomycin H/J was identified in the genome of Micromonospora sp. strain R45601, suggesting production of a chemical analog. LC-MS detected four peaks of interest, two of which are associated with mass-to-charge (m/z) values that do not correlate with any previously identified diazaquinomycin analogs. This analysis has identified at least two potentially novel anti-TB compounds, supporting continued investigation into sponge-associated marine actinomycetes for novel therapeutics.