Background: Infections of the bloodstream and sepsis are significant causes of morbidity and mortality, especially when complicated by multidrug resistance. Molecular diagnosis represents a tool for rapid identification of causative organisms with subsequent optimization of antimicrobial therapy; however, conventional blood culture still plays a critical role as a reference method for microbial identification and susceptibility testing. Methods: The study included 110 bloodstream isolates obtained from patients with suspected bacteremia and sepsis. Conventional blood culture was used for organism identification, incubation time analysis, and antibiogram testing, while multiplex PCR was used to screen for the most prevalent resistance determinants, namely NDM, OXA-48, KPC, VIM, mecA, and VanA/VanB genes. Bloodstream and non-bloodstream isolates from selected cases of sepsis were compared. Results: Klebsiella pneumoniae: 27 (24.5%), Staphylococcus aureus13 (11.8%), Escherichia coli8 (7.3%), Enterococcus faecium (7; 6.4%), Acinetobacter baumannii (8;7.3%), Enterococcus faecalis (6; 5.5%), and Pseudomonas aeruginosa (6; 5.5%) were the most prevalent species isolated from blood. The majority of cultures became positive within 20 hours from seeding, with 24 out of 97 (24.7%) positive between 0 and 10 hours and 55 out of 97 (56.7%) positive between 11 and 20 hours. The most prevalent resistance determinants were NDM, NDM+OXA-48, mecA, and VanA/VanB. Susceptibility testing revealed that carbapenems were largely ineffective against K.pneumoniae, while aztreonam/avibactam, tigecycline, fosfomycin, linezolid, vancomycin, and teicoplanin showed good activity against several strains of bacteria. Conclusion: The present findings demonstrate the potential utility of multiplex PCR as a rapid assay for detection of multidrug-resistant bloodstream pathogens and early optimization of antimicrobial treatment. At the same time, conventional blood culture remains an indispensable tool fo