Aureobasidium sp. strain TD-062, a microcolonial fungus, was isolated from the Thar Desert, India, and it’s aqueous and ethyl acetate extracts were evaluated for antimicrobial activity against clinical isolates of Pseudomonas aeruginosa (sputum), Escherichia coli (pus, blood, and urine), Klebsiella pneumoniae (urine), and Proteus. The ethyl acetate extract was analysed by gas chromatography-mass spectrometry (GC-MS) and examined for toxicity using ProTox-II software. Three compounds, selected based on toxicity and safety profiles, were analysed for molecular docking on BlaR1, a protein that induces antimicrobial resistance in bacteria. The ethyl acetate extract was shown to exhibit antimicrobial activity against clinical pathogens. GC-MS analysis showed that squalene, stigmasterol and delta-tocopherol had lower toxicity profiles. Molecular docking analyses demonstrated that stigmasterol exhibited the highest binding affinity (-8.9 Kcal/mol), compared to positive control clavulanic acid (-6.7 Kcal/mol), suggesting its potential as a potent BlaR1 inhibitor. These findings underscore the bioactive potential of Aureobasidium sp. TD-062 as a promising source of bioactive compounds to combat antimicrobial resistance. The identification of squalene, stigmasterol, and delta-tocopherol as lead compounds for drug development represents a significant advancement in the search for novel antimicrobial agents to address the growing global threat of antimicrobial resistance.