Staphylococcus aureus is a pathogenic bacterium capable of developing biofilms, leading to nosocomial infection and cross-contamination of foods. The current study was focused on the detection of adhesin genes, staphylococcal nuclease and hemolytic activities, and biofilm formation among the isolates of S. aureus from different sources. Fifteen adhesin genes (bap, bbp, clfA, clfB, cna, ebpS, fib, fnbA, fnbB, eno, icaAD, icaBC, sasG, sasC, pls) involved in S. aureus cell aggregation and biofilm accumulation were detected by polymerase chain reaction using specific primer. The activities of staphylococcal nuclease and hemolysis were analyzed by using toluidine blue-DNA agar and sheep blood agar for each strain. The ability of biofilm formation among different S. aureus strains was tested by using the glass tube method and microtiter-plate method. Our results showed the diversity of biofilm formation from different sources. Some isolates were strong biofilm producers; some were weak biofilm producers; and some were nonbiofilm producers. Staphylococcal nuclease and hemolysis seem to play a certain inhibitory role in biofilm formation. The adhesin genes varied among different S. aureus strains. The bap gene was not present in any strains. The bbp gene was only detected in one strain. The detection rates of other adhesin genes were as follows: clfB and sasG (100%); cna, eno, fib, and ebpS (93.75%); fnbA, icaAD, and icaBC (87.50%); fnbB (68.75%); sasC (31.25%); clfA (25%); and pls (12.50%), respectively. The variation between phenotypic and genotypic characterization may be due to the heterogeneity in the genetic origins. There was no direct correlation in distribution of adhesin genes and biofilm formation, which indicates that a single gene or subset of genes cannot be utilized as a biofilm indicator for morphology. Our results also indicated that biofilm formation might be affected by many factors, which brings new challenges to the prevention of this serious pathogen due to biofilm-related infection and contamination.