Many domestic cell culture laboratories are plagued by “black granules” contamination, which are commonly referred to as “nanobacteria” contamination in foreign countries, but the species of “black granules” has not been determined. “Black granules” were isolated from DMEM medium, and 16 S rDNA sequencing and VITEK-2 microbial identification system were used for the classification and identification of “black granules”. Antibiotic susceptibility assay and crystal violet staining technique were used to screen the antibiotics sensitive to “black granules” and their suitable concentrations for cell culture. We observed that “black granules” were spotted or flaky with an inverted microscope. The “black granules” was rod-shaped, with a length of 1 300-1 600 nm and a width of 400-500 nm under the transmission electron microscope, and “Brownian motion” was performed in situ under confocal laser scanning microscope. The results of 16 S rDNA sequencing and VITEK-2 microbial identification system showed that “black granules” was a new strain of Brevundimonas, and its sequences were 97.8% similar to Brevundimonas diminuta as well as 98.1% similar to Brevendomonas faecalis, respectively. Antibiotic susceptibility assay showed that “black granules” were sensitive to antibiotics chloramphenicol (25 mg/L), erythromycin (200 mg/L) and tetracycline (2.5 mg/L), but resistant to ampicillin (50 mg/L), puromycin (2 mg/L), penicillin-streptomycin (100×) or gentamicin-amphotericin B (100×). Crystal violet staining showed that the proliferation of “black granules” was inhibited by the chloramphenicol (25 mg/L), erythromycin (200 mg/L) or tetracycline (2.5 mg/L) in the DMEM medium, and the inhibition of “black granules” for cell proliferation were reduced. In summary, the “black granules” isolated and identified in our study was a new stain of Brevundimonas, and the inhibition of “black granules” for cell proliferation could be reduced by low concentration of chloramphenicol, erythromycin or tetracycline. Due to the limitations of this study, there may be other possibilities for the species of “black granules”. This study provides new research ideas and methods for species identification of “black granules” or nanobacteria and sterile culture of cells.