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Identification of anaerobic bacteria causing blood stream infections (BSI) is challenging. This study describes the epidemiology of anaerobic BSI at a tertiary care hospital and the performance of a rapid method for identification of anaerobic bacteria from blood cultures over three years, between June 2015 and June 2018. Short-term culturing is a low-cost user-friendly method and may be used for rapid identification of bacteria from positive blood cultures by matrix-associated laser desorption ionization time of flight (MALDI-TOF). Short-term culturing is performed after Gram staining on all positive blood culture bottles (BCBs) before 10:30 a.m. in our laboratory. Successful short-term cultures were defined as growth and reliable MALDI-TOF result in maximum 8 h after culture positivity. Data pertaining to unique anaerobic episodes and short-term cultures was collected retrospectively from our laboratory information system. Overall, during the three-year period, 692 unique anaerobic episodes (including Propionibacterium spp) were isolated. A total of 17 anaerobic bacterial genera were isolated in our laboratory, with 5 GNB genera and 12 GPB genera. The most prevalent bacteria were Bacteroides spp. 266/692 (38%) , Propionibacterium spp. 128/692 (18%), Clostridium spp. 103/692 (15%) , Fusobacterium spp. 34/692 (5%), and Actinomyces spp. 34/692 (5%). We performed short-term cultures on 270/564 (48%) clinically relevant episodes (excluding Propionibacterium spp) on chocolate agar plates. Growth within 8 h from culture positivity was detected in 33/270 (12%) short-term cultures. There were 22/33 (67%) gram negative (GNB) and 11/33 (33%) gram positive bacteria (GPB). Only two genera were identified: Bacteroides spp 22/33 (67%) and Clostridium spp 11/33 (33%). Thus, short-term culturing can function as a low-cost add-on to already existing protocols involving MALDI-TOF, where both GNB and GPB can be identified. • Bacteroides spp. (38%), Propionibacterium spp. (18%) and Clostridium spp (15%) were most common. • Short-term culture followed by MALDI-TOF MS can function as an add-on to conventional methods. • The method could identify 67% of Bacteroides spp. and 33% of Clostridium spp. within 8 h. [ABSTRACT FROM AUTHOR]