In an effort to overcome historical problems associated with the isolation of Bartonella species from animal and human blood samples, our laboratory developed a novel, chemically modified, insect-based, liquid culture medium (Bartonella alpha-Proteobacteria growth medium, BAPGM). In this study, we describe the isolation of non-Bartonella bacteria from aseptically obtained human blood and tissue samples that were inoculated into BAPGM pre-enrichment culture medium, and were obtained during attempts to define each individuals Bartonella infection status. After incubation for at least 7 days in liquid BAPGM, pre-enriched inoculums were sub-cultured onto a BAPGM/blood agar plate. Bacterial DNA was extracted from pooled plated colonies and amplified using conventional PCR targeting the 16S rRNA gene. Subsequently, amplicons were cloned, sequenced and compared to GenBank database sequences using the BLAST program. Regardless of the patient's Bartonella status, seventeen samples generated only one 16S rDNA sequence, representing the following genera: Arthrobacter, Bacillus, Bartonella, Dermabacter, Methylobacterium, Propionibacterium, Pseudomonas, Staphylococcus and bacteria listed as "non-cultured" in the GenBank database. Alkalibacterium, Arthrobacter, Erwinia, Kineococcus, Methylobacterium, Propionibacterium, Sphingomonas, and Staphylococcus were isolated from nine Bartonella-infected individuals. Co-isolation of Acinetobacter, Sphingomonas, Staphylococcus spp. and bacteria listed as "non-cultured" in the GenBank database was achieved for four samples in which Bartonella spp. were not detected. Despite the phylogenetic limitations of using partial 16S rRNA gene sequencing for species and strain identification, the investigational methodology described in this study may provide a complementary approach for the isolation and identification of bacteria from patient samples.