The worldwide increase in antimicrobial-resistant bacteria (ARB) presents a significant medical challenge and has emerged as one of the most critical concerns of our era. Antibiotic-resistant Klebsiella has emerged over the past few decades as a major public health threat. The prevalence of bacterial infections associated with biofilm formation is significant, accounting for approximately 65-80% of all cases. Given this scenario, this study investigates the prevalence of biofilm formation in Klebsiella isolated from surface waters and hospital wastewaters. In total, 77 samples were collected from surface waters in North Portugal, of which 33 Klebsiella (14 Klebsiella spp. and 19 K. pneumoniae strains) were isolated. Furthermore, 44 samples were collected from hospital wastewaters in Northern Portugal, of which 41 Klebsiella (25 Klebsiella spp. and 16 K. pneumoniae strains) were isolated. Biofilm production was evaluated using the microtiter plate assay. In surface waters, isolates of Klebsiella spp. and K. pneumoniae produced moderate biofilm biomass, with similar averages of biofilm formation percentages (83.1% and 86.9%, respectively). In K. pneumoniae strains, 65% (n=13) were confirmed as weak producers, 10% (n=2) were moderate, and 25% (n=5) were strong biofilm producers. Likewise, regarding Klebsiella spp., 35.7% (n=5) were weak producers, 57.1% (n=8) were moderate, and 7.1% (n=1) were strong biofilm producers. These were positive results, as the presence of biofilm-producing strains complicates infection treatment, given that antibiotics cannot eliminate the biofilm, only addressing the infection symptoms. With this, it is concluded that understanding and addressing biofilm formation is crucial for developing effective strategies for preventing and treating bacterial infections. Furthermore, detecting and controlling the spread of these and employing a One Health approach are vital for managing this problem effectively.