<p>The study’s primary objective was to develop Compritol-based lipid-polymer hybrid nanoparticles (LPHNs) of gentamicin (GTM) for transdermal delivery against aerobic bacteria. For this purpose, GTM-LPHNs were fabricated using a modified homogenization method, whereas optimization was carried out via the Design Expert’s Box-Behnken Design; these nanoparticles were then characterized via FTIR and TEM analysis. The optimized GTM-LPHNs were loaded in a transdermal patch and characterized along with the evaluation of in vitro drug release, ex vivo permeation, and antimicrobial assay. Optimized formulation exhibited a favorable particle size (99.36 ± 1.8 nm), polydispersity index (0.212 ± 0.006), zeta potential (− 25.45 ± 0.05 mV), and entrapment efficiency (76 ± 3.5%). The FTIR and TEM results further validated these nanoparticles. In vitro drug release from the GTM-LPHNs patch displayed a sustained release pattern (63.77 ± 2.4% in 72 h) compared to the GTM dispersion (95.75 ± 3.6% in 4 h). Additionally, ex vivo permeation studies confirmed the effect of permeation enhancer: menthol, with a significantly higher permeation flux (1500.23 ± 92.42 mcg/cm²/h) as compared to GTM-LPHNs without menthol (830.36 ± 6.98 mcg/cm²/h). Antimicrobial assay confirmed the antimicrobial potential of GTM-LPHNs against <i>S. aureus</i> and <i>E. coli</i>, with visible zones of inhibition recorded in the in vitro antimicrobial assay. The strength of this work lies in employing Compritol-based GTM-LPHNs to enhance the transdermal delivery of hydrophilic gentamicin, offering sustained release, improved permeation, and antibacterial efficacy against both Gram-positive and Gram-negative bacteria.</p> Graphical Abstract <p></p>