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Foodborne pathogens and biofilm formation pose a critical threat to food safety. Pseudomonas aeruginosa is a pathogen of interest in the food industry. High-power pulsed microwave (HPPM) is a non-thermal technology with proven efficacy towards bacterial inactivation. This study investigated the inactivation mechanisms and performance of different HPPM treatments (50, 100, 150, 200, and 250 Hz for 3, 5, 7, 9, and 11 min, respectively) to identify the optimum conditions to inactivate P. aeruginosa PAO1 biofilms. The results showed significant cell inactivation at increasing frequency/time (p < 0.05). Hence, HPPM treatment (200 Hz, 9 min) was selected following its efficacy and limited thermal sublethal effects to elucidate further the inactivation mechanisms against P. aeruginosa and its biofilm-forming ability. Cell viability, scanning (SEM) and transmission electron microscopy (TEM) results demonstrated severe morphological disruptions, cell disintegration, and cell lysis in the treated cells. From the confocal laser scanning micrographs (CLSM), biofilm thickness was calculated, showing a significant reduction after treatment (17.56 vs. 42.16 μm in the treated and untreated groups, respectively), indicating that HPPM effectively compromised the integrity of the biofilm envelope. This study outlines useful insights into better understanding HPPM's efficacy on microbial inactivation and biofilm proliferation in the food industry. • High-power pulse microwave (HPPM) demonstrated significant cell inactivation at increasing frequency/time (p < 0.05). • Treated cells demonstrated severe cell deformation, membrane shriveling, and cell lysis. • It showed a marked reduction in biofilm thickness than untreated cells (17.56 ± 1.51vs. 42.16 ± 1.23 μm). • This study provides useful insights into validating HPPM's efficacy against bacterial biofilm formation. [ABSTRACT FROM AUTHOR]