Bdellovibrio bacteriovorus is a gram-negative, obligate aerobic bacterium that preys on other gram-negative bacteria. A new method of isolating and cultivating host-independent B. bacteriovorus on nutrient-rich agar has been developed, and work with PCR and gel electrophoresis verified that the isolate is indeed B. bacteriovorus. Host-independent colonies produce a yellow compound that was formerly assumed to be a carotenoid synthesized to cope with cellular oxidative stress. However, extraction and characterization with NMR, FTIR, MS-MS, UV-visible spectrophotometry and cyclic voltammetry show several differences between this compound and typical carotenoids. Although the molecular mass of the compound (727 amu) is plausible for a carotenoid, the compound generated by B. bacteriovorus is highly saturated and has no aromatic functionalities. Its molar extinction coefficient is significantly lower than those for most carotenoids. It is readily reduced (E = -0.51 V), but its oxidation potential (E = 1.28 V) is much higher than that for typical carotenoids. These data give a nearly complete chemical structure and indicate that the yellow compound produced by B. bacteriovorus is not a carotenoid, but likely a biosurfactant. The biological role of biosurfactants will be discussed.