Klebsiella pneumoniae (K. pneumoniae) is an important multidrug-resistant (MDR) and biofilm producing pathogen that causes diverse infections in human and animals. This study aimed to evaluate the prevalence, antimicrobial resistance, and biofilm forming ability of K. pneumoniae isolates from diseased humans sputum and urine samples and mastitic cows milk samples in Zagazig city, Sharkia Governorate, Egypt. A total of 100 human and animal samples were collected and examined for the presence of Klebsiella species. The recovered isolates were identified by conventional bacteriological procedures and confirmed by the traditional PCR at the genus and species levels using gyrA, 16S-23S ITS and pehX genes, respectively. The confirmed K. pneumoniae isolates were subjected for testing their antimicrobial susceptibility and biofilm formation ability. The results revealed that 10 out of 50 human samples were positive for K. pneumoniae (20%); 28% (7/25) from sputum samples and 12% (3/25) from urine samples. Additionally, the prevalence rate of K. pneumoniae in milk samples from mastitic cows was 4% (2/50). PCR assays confirmed the 12 isolates as K. pneumoniae. The antimicrobial susceptibility results showed that K. pneumoniae isolates had high resistance rates to ampicillin and ampicillin/clavulanate (100% each) with multiple antibiotic resistance (MAR) index of 0.083, followed by azithromycin, cefepime, and trimethoprim/sulphamethoxazole (91.7% each). Meanwhile, these isolates revealed high sensitivity levels to chloramphenicol (83.3%) with MAR index of 0.014. Furthermore, 91.7% of K. pneumoniae isolates were able to produce the biofilm, where 8.3% was a non-biofilm producer. Out of 11 K. pneumoniae isolates, 6 (54.5%), 3 (27.3%) and 2 (18.2%) were moderate, strong and weak biofilm producers, respectively. The present study emphasized the high prevalence of MDR and biofilm producing K. pneumoniae isolates in human sputum and urine samples. Therefore, more attentions should be taken against antimicrobials usage and for providing new antibiofilm approaches against Klebsiella species biofilms.