Clinical significance of a single positive blood culture bottle (SPBCB) with is unclear. We aimed to assess the significance of an SPBCB by looking at the associated outcomes. We performed a retrospective, multicenter study of patients with an SPBCB with using data collected from both electronic health records and the clinical microbiology laboratory. Overall, 534 patients with bacteremia were identified and 118 (22.1%) had an SPBCB. Among cases with an SPBCB, 106 (89.8%) were classified as clinically significant whereas 12 (10.2%) were considered contaminated or of unclear significance. A majority (92.4%) of patients received antibiotic therapy, but patients with clinically significant bacteremia were treated with longer courses (25.9 vs 5.7 days,  < .001). Significant differences in both frequency of echocardiography (65.1% vs 84.6%,  < .001) and infective endocarditis diagnosis (3.8% vs 14.2%,  = .002) were seen in those with an SPBCB compared to those with multiple positive bottles. A longer hospital length of stay and higher 90-day, 6-month, and 1-year mortality rates were seen in patients with multiple positive blood culture bottles. An SPBCB with was common among our patients. While this syndrome has a more favorable prognosis as compared to those with multiple positive blood cultures, clinicians should remain concerned as it portends a risk of infective endocarditis and mortality.