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An increase in the antibiotic resistance among Enterococcus faecium strains has been observed worldwide. Moreover, this bacteria has the ability to produce several virulence factors and to form biofilm that plays an important role in human infections. This study was designed to compare the antibiotic resistance and the prevalence of genes encoding surface protein (esp), aggregation substance (as), surface adhesin (efaA), collagen adhesin (ace), gelatinase (gelE), and hialuronidase (hyl) between biofilm-producing and non-producing E. faecium strains. Therefore, ninety E. faecium clinical isolates were tested for biofilm-forming ability, and then were assigned to two groups: biofilm-positive (BIO(+), n =70) and biofilm-negative (BIO(-), n = 20). Comparison of these groups showed that BIO(+) isolates were resistant to β-lactams, whereas 10% of BIO(-) strains were susceptible to ampicillin (statistically significant difference, p = 0.007) and 5% to imipenem. Linezolid and tigecycline were the only antibiotics active against all tested isolates. Analysis of the virulence factors revealed that ace, efaA, and gelE genes occurred more frequently in BIO(-) strains (ace in 50% BIO(+) vs. 75% BIO(-); efaA 44.3% vs. 85%; gelE 2.9% vs. 15%, respectively), while hyl gene appeared more frequently in BIO(+) isolates (87.1% BIO(+) vs. 65% BIO(-)). These differences were significant (p < 0.05). We concluded that BIO(+) strains were more resistant to antibiotics than BIO(-) strains, but interestingly, BIO(-) isolates were characterized by possession of higher virulence capabilities.