Background Febrile neutropenia often occurs as a serious complication in patients receiving chemotherapy. It is regarded as an oncologic emergency due to its high morbidity and mortality, necessitating the prompt initiation of empirical antibiotic therapy. While gram-negative bacteria have historically been the primary cause of bloodstream infections in febrile neutropenic patients, recent shifts in bacterial epidemiology and antimicrobial resistance patterns underscore the need for regular evaluation of local data to inform treatment strategies. Methods This retrospective cohort study was conducted in the Department of Hematology at Victoria Hospital, Kirkcaldy, from January to December 2024. It analyzed blood culture results, antibiotic use, chemotherapy regimens, patient characteristics, and microbial susceptibility patterns. A total of 63 patients with an absolute neutrophil count of less than 0.5 were selected through a simple convenience sampling method. Demographic data were collected from ward admission records using a standardized data collection sheet. Blood culture results were retrieved from the laboratory information management system and analyzed using IBM SPSS Statistics for Windows, Version 23.0 (Released 2015; IBM Corp., Armonk, NY, USA). Results Among the 63 patients, 20 (32%) had at least one positive blood culture, with a male-to-female ratio of 3:2. Gram-positive bacteria accounted for the majority of isolates (11; 55%), followed by gram-negative bacteria (eight; 40%), and mixed growth in one case (5%). was the most common isolate (five; 25%), followed by species and (four; 20% each). was identified in 3 cases (15%). The most common pathogens associated with line infections were (three; 25%), (three; 25%), and species (four; 33%). A statistically significant association was observed between the presence of a central venous line and the occurrence of bacteremia (p < 0.05). Most gram-negative isolates were susceptible to beta-lactam antibiotics and gentamicin, while gram-positive organisms showed high sensitivity to vancomycin and linezolid. Although gram-positive bacteria were the most frequently isolated organisms overall, was the single most common isolate and demonstrated susceptibility to the current first- and second-line antibiotics used in the treatment of neutropenic sepsis. Conclusions The findings of this study are broadly consistent with existing literature on bloodstream infections in neutropenic cancer patients, particularly in terms of the microbial spectrum and the association with central venous lines. However, variations in pathogen prevalence and antibiotic susceptibility may reflect local epidemiology or specific cohort characteristics. These results highlight the need to continually assess and update antibiotic guidelines to mitigate the development of drug-resistant strains, ensure adherence to current protocols, and improve the accuracy of empirical antibiotic prescribing.