The presence of microorganisms in the bloodstream can result in severe, potentially life-threatening conditions, which are a significant cause of morbidity and mortality worldwide. The rise of antimicrobial-resistant strains further exacerbates these outcomes. However, the data concerning bacterial profiles and resistance to antimicrobials, particularly against extended-spectrum beta-lactams and carbapenems, are limited. Aimed to characterize pathogens isolated from positive blood cultures, including bacterial profiles and antibiotic susceptibility patterns, and to identify predictors of blood culture positivity in septicemia-suspected patients at the University of Gondar Comprehensive Specialized Hospital, Northwest Ethiopia. A hospital-based cross-sectional study was conducted from February 15 to May 30, 2023. The study involved 341 patients suspected of having septicaemia who were selected consecutively through a convenience sampling technique. Blood samples were collected aseptically from each patient (10 ml from adults, 5 ml from children, and 1 ml from neonates) and inoculated into bottles containing tryptic soy broth in volumes appropriate for the patient's age. The samples were incubated at 35-37 °C for up to 7 days to detect bacterial growth. Positive blood cultures were subcultured onto various media, including chocolate agar, blood agar, modified Thayer-Martin agar, MacConkey agar, and mannitol salt agar, and incubated again at 35-37 °C for 24 h. The suspected bacteria were identified on the basis of colony morphology, Gram staining, and biochemical tests. Antimicrobial susceptibility testing was conducted via both the Kirby-Bauer and modified Kirby-Bauer disk diffusion methods. Resistance to methicillin, extended-spectrum beta-lactams, and carbapenems was determined via the cefoxitin disc test, combined-disk diffusion method, and modified carbapenem inactivation method, respectively. The data were entered into Epi-Data version 4.6 and analysed via SPSS version 25. Binary logistic regression analyses were employed to identify factors associated with bloodstream infections (BSI), with a P value of less than 0.05 considered statistically significant. Out of 341 patients suspected of septicemia, 196 (57.5%) were male and 145 (42.5%) were female, with a mean (± SD) age of 16.5 (± 7.5) years. Bloodstream infection was identified in 87 (25.5%) patients (95% CI: 21.1-30.4). Among these positive cases, 67 (77%) were from ward patients, while 20 (23%) were from those visiting outpatient departments. The primary gram-positive bacteria identified included S. aureus 27 (31.0%), CoNS 14 (16.1%), S. viridans 8 (9.2%), and S. agalactiae 4 (4.6%). The gram-negative isolates were predominantly K. pneumoniae 11 (12.6%), followed by E. coli 9 (10.3%), E. cloacae 6 (6.9%), Acinetobacter spp. 3 (3.5%), N. meningitidis 3 (3.5%), and P. aeruginosa 2 (2.3%). Methicillin resistance was detected in 17/27 (63.0%) S. aureus strains and 2/14 (14.3%) CoNS strains. Multidrug resistance was detected in 63/87 (72.4%, 95% CI: 67.2-84.7%) of the isolates. Extended-spectrum beta-lactamase and carbapenemase production were observed in 12/31 (38.7%) and 5/31 (16.1%) of isolates, respectively. The factors associated with BSI were the presence of wounds and burns (AOR = 2.103, 95% CI: 1.365-3.241, P = 0.041), length of hospital stay (≥ 5) (AOR = 2.209, 95% CI: 1.122-4.347, P = 0.022), and prior medical procedures (AOR = 1.982, 95% CI: 1.125-3.492, P = 0.018). Bloodstream infection was identified in 25.5% of suspected septicemia cases, with multidrug-resistant bacteria present in 72.4% of isolates. Gram-positive bacteria, particularly S. aureus, and gram-negative bacteria like K. pneumoniae and E. coli were predominant. High rates of methicillin, beta-lactam, and carbapenem resistance were observed, emphasizing the magnitude of antimicrobial resistance. Risk factors such as wounds, extended hospital stays, and prior medical procedures significantly increased the likelihood of culture positivity. This suggests the need for regular antimicrobial susceptibility testing to guide antibiotic selection and track resistance trends, proper wound care and medical device usage to reduce the risk of BSI in healthcare settings.