Background: Borrelia burgdorferi, the causative agent of Lyme disease, releases outer membrane vesicles (OMVs) that may contribute to infection and modulate the host immune response. Although interest in OMVs is growing, few studies have systematically compared methods for isolating OMVs from B. burgdorferi. Methods: In this study, we evaluated two OMV isolation techniques—standard ultracentrifugation and an ion-exchange chromatography-based ExoBacteria™ kit—and examined how serum supplements (rabbit serum vs. exosome-depleted fetal bovine serum, ED-FBS) influence Bb-OMV yield and composition. Gene expression profiles were assessed using RT-PCR, and specific protein content was identified by Western blot analyses. To assess the ability of Bb-OMVs to interact with host cells, Bb-OMVs were co-cultured with MDA-MB-231 triple-negative breast cancer cells. Results: Transmission electron microscopy confirmed that both methods produced spherical Bb-OMVs with intact membrane bilayers. Ultracentrifugation generated larger vesicles (15–180 nm), while the ExoBacteria™ kit yielded smaller vesicles (<50 nm) with a higher double-stranded DNA (dsDNA) content, and protein levels were similar across samples. Cultures grown with rabbit serum produced more Bb-OMVs and had cleaner backgrounds in the TEM images than those grown with ED-FBS. All Bb-OMV samples lacked intracellular markers (DnaK and 16S rRNA) and consistently expressed the outer surface protein OspA, confirming high purity. All isolated Bb-OMVs were taken up by the cells, as indicated by OspA expression, without detectable 16S rRNA, confirming vesicle internalization without bacterial contamination. Conclusions: These findings indicate that isolated OMVs are biologically active and capable of interacting with mammalian cells, highlighting their potential role in host–pathogen interactions and the broader relevance of OMVs in studying bacterial modulation of mammalian cell behavior. Overall, both isolation methods produced high-quality OMVs, with ultracentrifugation yielding slightly more pure vesicles, emphasizing the importance of selecting appropriate isolation methods and culture conditions for functional OMV studies.