Antibiotics are an important resource in modern medicine used to treat serious infections and enable a wide array of vital medical interventions including surgery and cancer chemotherapy. However, due to the increasing prevalence of antibiotic resistant pathogens, many clinically useful antibiotics are being rendered ineffective with too few new antibiotics in development to combat them. With highly diverse chemistry and bioactivity exquisitely shaped by evolution, natural products provide an unrivaled source of antibiotic compounds that is impossible to reproduce instinctively in the laboratory. The armeniaspirols are polyketide natural products with a unique spiro-[4.4]non-8-ene core that were isolated from Streptomyces armeniacus and were shown to be active against drug-resistant Gram-positive bacteria. Promisingly, in vitro resistant Staphylococcus aureus strains could not be readily obtained even after thirty serial passages under sub-lethal doses. Herein, we decipher the mechanism of action for this structurally unprecedented natural product antibiotic in the Gram-positive model organism Bacillus subtilis. Through chemical proteomics with an armeniaspirol-inspired activity-based probe, quantitative proteomics, biochemical assays, and microscopy, we show that armeniaspirol is a competitive inhibitor of the AAA+ proteases ClpXP and ClpYQ. Armeniaspirol represents the first known natural product inhibitor of ClpP, a highly coveted target due to its prominent role in bacterial virulence. Using overlapping proteomic fingerprints of armeniaspirol-treatment with ΔclpQ and ΔclpP deletions in B. subtilis, inhibition or deletion of these proteases appears to dysregulate key proteins involved in cell division, including FtsZ, DivIVA, and MreB. The dual ClpXP and ClpYQ inhibition is responsible for armeniaspirol’s potent antibiotic activity and this unique pharmacology makes it a promising candidate for antibiotic development. Several armeniaspirol-inspired analogs were generated as part of a medicinal chemistry study and evaluated for antibiotic activity towards a panel of clinically relevant Gram-positive pathogens. As a result, we identify three exciting armeniaspirol analogs with improved antibiotic activity. Lastly, the foundation for elucidating the ClpYQ degradome is developed. Our proteomic fingerprint of the B. subtilis ΔclpQ deletion strain generated some of the first insights into potential substrates of the ClpYQ protease. As a largely uncharacterized AAA+ protease implicated in the mechanism of action of armeniaspirol, we pursued a previously established acyl-intermediate covalent trapping strategy to characterize the ClpYQ-substrate complexes in B. subtilis cell lysate. Through unnatural amino acid incorporation using an evolved tRNA/aminoacyl-tRNA synthetase pair, the N-terminal active site serine of ClpQ is substituted with a photocleavable precursor that generates 2,3-diaminopropionic acid. While we were successful in synthesizing the photocleavable precursor, initial experiments to incorporate this unnatural amino acid in ClpQ expression proved unsuccessful, leading us to outline necessary control experiments for future endeavours. Ultimately, the covalently trapped substrates will be identified by LC-MS/MS, where we expect to identify key divisome and elongasome proteins in corroboration with the armeniaspirol mechanism of action study.