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Objectives Blood stream infections (BSIs) are the main cause of morbidity and mortality in children worldwide. The present study was carried out to determine the prevalence of BSI with a focus on the identification of the causative agent of BSI, and to further evaluate the antibiotic susceptibility profile of the causing bacterial pathogens. Methods A cross-section study was carried out at the tertiary care hospital in Peshawar, Pakistan from January to December, 2018. Blood samples were collected in BACTECTM bottles and standard microbiological protocols were applied for the isolation and identification of bacterial strains. The antibiotic susceptibility tests were performed using disc diffusion method as per the 2014 guideline of Clinical Laboratory Standard Institute (CLSI). Results Of 567 blood cultures in total, 111(19.6％) were positive for BSI. Male children were 64 ％ (71/111) and female children were 36％ (40/111). For the causative predominant group of microorganisms, Gram-negative bacteria were identified in 79 (71.1％) isolates, and Gram-positive bacteria in 32 (28.9％) isolates. The common bacteria of isolates were S. typhi (n=35, 31.5％), E. coli (n=19, 17.1％), S. aureus (n=18, 16.2％), K.pneumonia (n=12, 10.8％), and Enterococcus species (n=7, 6.3％). The 36.7％ (29/79) isolates of Gram-negative bacteria were ESBL producers, and 61.1％ (11/18) of S. aureus isolates were methicillin resistant. Overall, 72.9％ isolates were multidrug resistant.Conclusions Gram-negative bacteria were the main cause of pediatric BSIs, where the predominant microorganism was S. typhi. Remarkably, majority of the S. typhi isolates were resistant to ciprofloxacin.