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Introduction. Anaerobic bacteria are important etiological agents of infections linked to human morbidity and mortality. Nevertheless, the limited development of most Costa Rican health care facilities with respect to the isolation and manipulation of anaerobic bacteria prevents the compilation of precise data on the prevalence of this sort of bacteria in the country. Objective. To assess the occurrence of anaerobic bacteria in clinical samples collected in a regional hospital in Costa Rica. Material and Methods. During 18 months, anaerobic bacteria were cultivated from diverse aspirates following a standardized protocol for sample collection and isolation developed for this study. Results. Forty seven anaerobic bacteria were isolated from 22 of 46 samples analyzed (48% of prevalence). While 25 of these organisms exhibited Gram-negative staining (53%), 22 isolates were characterized as Gram-positive (47%). Mostbacteria were allocated to the genus Bactero ides (42%), followed by Eggerthella (13%) and Propionibacterium (11%). A limited number of isolates was identified as Prevotella sp., Bifidobacterium sp., Porphyromonas sp., or Fusobacterium sp. Discussion. Although traditional procedures for sample collection and isolation of anaerobic bacteria are painstaking, a diversity of anaerobic species, including very strict and fastidious species, was isolated with the practical method applied in the current investigation. On account of this remark, we urge others to implement our protocol in their health care facilities, as this would likely help to define more precisely the role that anaerobic bacteria play in clinical infections. [ABSTRACT FROM AUTHOR]