The purpose of this paper is to define guidelines to interpret positive blood cultures (BCs) to distinguish bloodstream infection (BSI) from contamination in BCs drawn with a single venipuncture. During a 2-year period, each positive BC set (comprising six bottles from a single venipuncture) was prospectively categorised by clinicians, bacteriologists and hospital epidemiologists as BSI or contamination. For each case, the number of positive bottles per set, results from Gram staining and microorganism identification were analysed in order to define interpretation guidelines. We analysed 940 positive BC sets. The BSI rate in monomicrobial BC sets was positively correlated with the number of positive bottles. The positive predictive value was 88% with one and 100% with ≥2 positive bottles for Escherichia coli; 100% for Staphylococcus aureus, Pseudomonas and Candida spp., regardless of the number of positive bottles; 3.5%, 61.1%, 78.9% and 100% for coagulase-negative staphylococci (CoNS) with one, two, three and ≥4 positive bottles, respectively. Using a single-sampling strategy, interpretation guidelines for monomicrobial positive BCs are based on the number of positive bottles per set, results from Gram staining and microorganism identification: ≥4 positive bottles (≥2 with Gram-negative bacilli) always led to a diagnosis of BSI. The CoNS BSI rate positively correlates with the number of positive bottles.