Background: Bloodstream infections (BSI) in immunocompromised patients suffering from hematological malignancies continue to be an essential cause of morbidity and mortality. Methicillin-resistant Staphylococcus aureus (MRSA)-related BSI in patients with febrile neutropenia (FN) is a life-threatening bacterial infection and extremely challenging to treat. Methods: Blood samples were collected from febrile neutropenic patients. Conventional blood culture and direct PCR identification of 16S rRNA, mecA, femA, nuc, and lukS genes were performed for detection of MRSA. Antibiotic sensitivity profiles of the isolates were investigated using disc diffusion and minimum inhibitory concentration methods. Results: Among 24 positive blood cultures isolates, MRSA (12/24, 50%) was the predominant bacteria followed by coagulase negative staphylococci (CoNS) (6/24, 33.3%). All MRSA isolates were resistant to cefoxitin (MIC ≥ 8 μg/ml), and oxacillin (MIC ≥ 4 μg/ml) and harbored mecA gene. 10/12 MRSA isolates were vancomycin resistant (VRSA) (MIC ≥ 16 μg/ml). PCR for 16S rRNA and mecA genes yielded positive results in 14 negative blood culture samples. Conclusions: We cannot rely on blood culture as a reliable method for BSI diagnosis in patients with FN. 16S rRNA and characteristic MRSA genes PCR showed important role for diagnosis of culture-negative MRSA BSI particularly in patients with preceding prophylactic or empirical antibiotics.