The present study is the first molecular epidemiological study in Lithuania that covers the evaluation of resistance of both Acinetobacter spp. and E. coli to various antibiotic groups, as well as virulence gene identification, phylogenetic isolation, application, and analysis of different genotyping methods. This study describes 194 Acinetobacter spp. and 256 multidrug-resistant E. coli isolates from hospitalised patients diagnosed with sepsis. Antibiotic susceptibility genes, which have been minimally studied in Lithuania, have been characterised in this study. These include the OXA group β-lactamases, metallo-β-lactamases, and carbapenemases. Resistance to aminoglycoside-, quinolone-, and polymyxin-encoding genes have not been extensively studied in Europe and have never been identified in Lithuania prior to this study. Similarly, regulatory genes for efflux pumps, which are increasingly being detected worldwide, were identified in Lithuania for the first time. Genes encoding virulence factors responsible for the invasion and pathogenesis of ExPEC strains in the human body, have also been identified for the first time in Lithuania. Phylogenetic groups of E. coli isolates from different Lithuanian hospitals were also identified in this study, which further supplemented the results of previous studies in Lithuania. Moreover, it was determined that 45.9% of all Acinetobacter spp. isolates exhibited an β-lactams resistance gene combination of blaOXA subgroup-3-blaOXA subgroup-1-blaOXA51-blaOXA sugroup-2-blaOXA-subgroup-4-blaVIM-1-blaTEM-92. Moreover, the most common resistance gene combination in E. coli isolates was tetA-strB-sul2-blaTEM-blaNDM-strA-fosA-blaAIM-sul3-aadA-blaCTX-M-9, which caused resistance to β-lactams, aminoglycosides, sulphonamides, fosfomycin, and tetracyclines. The most common virulence gene combination was fuyA-fimH-iroN, where fuyA and iroN encode siderophores and fimH is responsible for bacterial adhesion to host cells. Phylogenetic group determination was performed for all E. coli isolates. The isolates belonged to four phylogenetic groups: A, B1, B2, and F. Group A isolates were detected at a significantly higher frequency (79.3% of all isolates) than isolates of groups B1 (0.8%), B2 (15.6%), and F (4.3%). Acinetobacter spp. and E. coli isolates from different Lithuanian hospitals were described in detail by different genotyping methods, with a strong focus on the analysis of the genotypic profiles and the investigation of possible associations between the isolation year and the particular hospital. BOX-PCR genotyping analysis was performed on all 194 Acinetobacter spp. isolates, a total of 191 BOX-PCR profiles were identified, which were separated into six clusters. In total, 235 BOX-PCR profiles of E. coli isolates were obtained, where all profiles were separated into 14 genotypic clusters. Moreover, it was observed determination of resistance and virulence genes to genotyping profiles and results showed potential changes in these genes. Also, at this work MLVA genotyping was used to describe the VNTR profiles of studied bacteria and results could be used for further analysis of future distribution Acinetobacter spp. and E coli multidrug resistant strains and their potential treatment strategies in different healthcare institutions.