Lysine acylation is a post-translational modification (PTM) conserved in all domains of life and is essential for regulating diverse biological processes. Traditional methods for investigating acylation rely on anti-acyl-lysine-antibodies, which are costly, time-consuming, and often exhibit variable affinity. To remedy these pitfalls, we developed an antibody-free method for bacterial acylome enrichment using bioorthogonal click chemistry coupled with tandem mass spectrometry. We applied this approach to the pathogens Pseudomonas aeruginosa and methicillin-resistant Staphylococcus aureus (MRSA) to understand the regulatory role of acylation in each organism. We characterized the acetylome, propionylome and butyrylome in P. aeruginosa UCBPP-PA14 and the acetylome and propionylome in MRSA. Comparative analyses revealed distinct PTM dynamics showing that acylation regulated a wide range of cellular functions, including metabolism, antibiotic resistance, virulence, and stress response. This work establishes the first antibody-free enrichment method for defining bacterial acylomes and provides new insight into global lysine acylation networks in pathogenic bacteria.