Pseudomonas aeruginosa and Klebsiella pneumoniae are notorious for their resistance to antibiotics and propensity for biofilm formation, posing significant threats to human health. Epsilon-poly-L-lysine (ε-PL) emerges as a naturally occurring antimicrobial poly(amino acid), which positions it as a prospective agent for addressing challenges linked to multidrug resistance. ε-PL symbolizes a promising avenue in the pursuit of efficacious therapeutic strategies and warrants earnest consideration within the realm of clinical treatment. Thus, our objective was to determine the antibiotic susceptibility profiles of 38 selected P. aeruginosa and ESBL-producing K. pneumoniae clinical isolates and determine the ability of ε-PL to inhibit biofilm formation. After PCR analysis, detection of genes related to β-lactamases was observed among the selected isolates of P. aeruginosa [blaSPM (35.7%), blaKPC (35.7%), blaSHV (14.3%), blaCTX-M (14.3%), blaOXA (14.3%), blaTEM (7.1%), blaPER (7.1%), blaVIM (7.1%), and blaVIM-2 (7.1%)] and K. pneumoniae [blaCTX-M (91.7%), blaTEM (83.3%), blaKPC (16.7%), blaNDM (12.5%), and blaOXA (4.2%)]. The results of testing the activity of ε-PL against the clinical isolates showed relatively high minimum inhibitory concentrations (MICs) for the P. aeruginosa (range: 8–64 µg/mL) and K. pneumoniae isolates (range: 16–32 µg/mL). These results suggest the need for prior optimization of ε-PL concerning its viability as an alternative to antibiotics for treating infections caused by P. aeruginosa and K. pneumoniae of clinical origin. It is noteworthy that, in the context of a low antibiotic discovery rate, ε-PL could play a significant role in this quest, considering its low toxicity and the unlikely development of resistance. Upon exposure to ε-PL, P. aeruginosa and K. pneumoniae isolates exhibited a reduction in biofilm production, with ε-PL concentration showing an inverse relationship, particularly in isolates initially characterized as strong or moderate producers, indicating its potential as a natural antimicrobial agent with further research needed to elucidate optimal concentrations and application methods across different bacterial species. Further research is needed to optimize its use and explore its potential in various applications.