<abstract> <p>Antibiotic resistance is one of the most important global healthcare challenges and is responsible for the mortality of millions of people worldwide every year. It is a crisis attributed to misuse of antibiotics and a lack of new drug development. Actinomycetes constitute a group of Gram-positive bacteria known for their distinctive high guanine-cytosine (G+C) content in their genomic DNA. These microorganisms are widely recognized for their capability to generate a wide range of secondary metabolites with diverse biological activities. These versatile microorganisms are ubiquitous in diverse ecosystems, including soil, freshwater, marine sediments, and within the bodies of insects. A recent study has demonstrated that social insects, such as ants, host a diverse array of these bacteria. In this study, we involved the isolation and characterization of a total of 72 actinomycete strains obtained from 18 distinct ant species collected from various regions across Thailand. Utilizing 16S rRNA gene analysis, these isolated actinomycetes were classified into four distinct genera: <italic>Amycolatopsis</italic> (2 isolates), <italic>Micromonospora</italic> (1 isolate), <italic>Nocardia</italic> (8 isolates), and <italic>Streptomyces</italic> (61 isolates). Among the <italic>Streptomyces</italic> strains, 23 isolates exhibited antimicrobial activity against a panel of Gram-positive bacteria, including <italic>Bacillus subtilis</italic> ATCC 6633, <italic>Staphylococcus epidermidis</italic> ATCC 12228, <italic>Staphylococcus aureus</italic> ATCC 25923, <italic>Kocuria rhizophila</italic> ATCC 9341, and Methicillin-resistant <italic>Staphylococcus aureus</italic> (MRSA) DMST 20646. Additionally, two isolates displayed antifungal activity against <italic>Candida albicans</italic> TISTR 5554. Based on 16S rRNA gene sequence similarity studies, these two isolates, ODS25 and ODS28, were demonstrated to be closely related to <italic>Streptomyces lusitanus</italic> NBRC 13464^T (98.07%) and <italic>Streptomyces haliclonae</italic> DSM 41970^T (97.28%), respectively. The level of 16S rRNA gene sequence similarity below 98.65% cutoff indicates its potential as a novel actinomycete species. These findings underscore the potential of actinomycetes sourced from ants as a valuable reservoir of novel antimicrobials.</p> </abstract>