Abstract: In this study, Cronobacter spp. food isolates were identified, and the biofilm formation ability, antimicrobial susceptibility and virulence genes were investigated. All 129 food samples were collected from food markets and food stands from Chengdu city in 2016.Cronobacter spp. were isolated and identified by DFI selective medium and 16S rRNA sequencing analysis. The 96-well microplate and tube test were used to detect biofilm formation ability. The influence of temperature on the biofilm formation was also explored. The antimicrobial susceptibility of Cronobacter spp. isolates to 18 antibiotics was detected by disk diffusion method. The virulence genes (cpa, hly, sip, and ompX) were detected by PCR. The results showed 43 Cronobacter spp. strains were identified from 129 food samples. The Cronobacter spp. isolation rate was 33.3%. The biofilm formation rate was 90.7% and the temperature had a significant effect on bacterial biofilm formation. The detection rate of ompX gene in 43 Cronobacter spp. strains was 100%;cpa was 13.9%, hly was 11.6%, and the sip gene was not detected in all isolates. The drug resistance rates to penicillin, clindamycin, vancomycin, oxacillin and bacitracin B for all 43 Cronobacter were 100%. The drug resistance rate to rifampicin was 97.7%. For erythromycin, the resistance rate was 7%. All the Cronobacter food isolates were sensitive to ciprofloxacin, gentamicin, tetracycline, chloramphenicol, imipenem, sulfamethoxazole, nitrofurantoin, cefoxitin, streptomycin, amikacin, and ofloxacin, completely. The study indicated Cronobacter spp. food isolates had better biofilm formation ability and had some resistance to most antibiotics. The food safety were threatened by Cronobacter spp. isoaltes with virulence genes.