Two new commercially available universal rRNA gene PCR plus sequencing tests, SepsiTest and universal microbe detection (UMD; Molzym, Bremen, Germany), were evaluated using blood specimens and heart valves from 30 patients with suspected infectious endocarditis (IE). The sensitivity of PCR (85%) was nearly twice as high as that of culture (45%), which in 10/20 IE cases presumably stayed negative as a consequence of growth inhibition of the pathogens by antibiotics. Further, PCR provided the basis for reclassification of 5/10 non-IE cases into IE cases. Culture-negative infections were identified by PCR, including single infections due to streptococci and Gram-negative bacteria (Escherichia coli, Haemophilus parainfluenzae) and mixed infections involving two Gram-positive bacteria or Candida spp. with Gram-positive bacteria. The new commercial tests proved to be of value for the rapid diagnosis of IE, particularly in cases of culture-negative infections. Issues regarding the feasibility of these tests for routine use are discussed.