Introduction: The choice of antimicrobial treatment for septicemia is usually empirical and based on health knowledge of local antimicrobial activity patterns of the most prevalent bacteria causing such bloodstream infections. The present study aimed to investigate the prevalence of bacterial pathogens causing bacteremia and their antimicrobial resistance profiles in hospitalized subjects. Materials and Methods: This cross sectional study was done at a major referral hospital, Faghihi Hospital, Shiraz, Iran. We examined 1262 positive blood cultures from 6300 specimens over a period of twelve months from September 2015 to September 2016. Results: Bacterial strains were isolated from 20% of blood cultures. The identified gram-negative bacteria were Alcaligenes (29.6%), Escherichia coli (7%), Klebsiella (3.7%), Acintobacter baumannii (3.6%), Pseudomonas (2%), Enterobacter (1.2%) and Brucella (1%). Of gram positive strains Staphylococcus aureus (27.2%) Staphylococcus epidermidis (11.4%) Non-hemolytic streptococci (4.6%), Diphtroids (4%), Enterococci (3.6%), and Micrococci (0.6%), were the most frequent isolates. Imipenem, Piperacilin/ tazobactam, Gentamicine and Amikacin were the most effective antibiotics against gram negative agents. Vancomycin, Rifampin, Cephalotin and Cefazolin were the most active antibiotics against gram negative bacterial agents. Conclusion: Resistance to majority of the antimicrobial agents for several pathogens implicated in bloodstream infections, particularly in gram-negative bacteria which cause complications in treatment of septicemia. Since S. epidermidis and Alcaligenes are normal flora of human’s skin and body, contamination of needle during blood sampling must be considered in such positive results. So we recommend considering better infection control precautions in blood sampling in the hospital.