Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (M.tb). According to the World Health Organization (WHO), TB is one of the top 10 causes of death worldwide and the leading cause of death from a single infectious agent. There were an estimated 1.2 million TB deaths among HIV negative people in 2019 and an additional 208 000 deaths among HIV positive people. Although there are drugs that can cure TB, drug-resistant TB continues to be a public health threat. In 2019, there were about half a million new cases of rifampicin-resistant TB (of which 78% had multidrug-resistant TB). Novel drugs need to be isolated and developed to combat this spread of drug resistance. Historically, natural product screening has been an effective tool in anti-tuberculosis drug discovery. Examples include rifampicin, derived from rifamycin which was originally isolated from the bacterium Amycolatopsis rifamycinica and streptomycin originally isolated from Streptomyces griseus. Both these bacteria belong to the phylum Actinobacteria. Due to this history, filamentous actinobacteria were selected for screening of novel antimycobacterial compounds. However, the focus in this investigation was on species from understudied genera, the so-called “rare actinobacteria” that have not been extensively explored for antimycobacterial compounds – to increase the likelihood of discovering novel antimycobacterial compounds. The actinobacteria were assessed for antimycobacterial properties using Mycobacterium aurum A+ by the overlay method. M. aurum has been shown to be a suitable indicator for Mycobacterium tuberculosis, is safer to work with, and more amenable to high-throughput screening. Once active strains were identified by the overlay method, the zone of inhibition was excised, and the compounds were extracted using ethyl acetate. The extracts were analysed with a high-resolution mass spectrometer, and their mass spectral data were analysed using Global Natural Products Social (GNPS) molecular networking methodology. Molecular networking allowed for the dereplication of known compounds early in the drug discovery process. Strains Amycolatopsis circi S1.3T , Actinomadura strain M2 grown in ISP2 medium and Kribbella speibonae SK5 produced active fractions. These compounds could not be matched to any known compound in the GNPS database and, therefore, could be novel antimycobacterial compounds.