Background: Data regarding differences in biofilm formation among urinary isolates of enterococci causing nosocomial infection versus asymptomatic colonisation is lacking. Conflicting data are available for the role of enterococcal surface protein (esp) gene in the development of enterococcal biofilms. Materials and Methods: A total of 50 (25 each of asymptomatic bacteriuria and urinary tract infection (UTI) isolates were collected from admitted patients who had nosocomial acquisition of enterococci in urine culture (≥105 cfu/ml). Biofilm assay was done by the quantitative adherence assay. Screening for esp gene was carried out by polymerase chain reaction, and confocal laser scanning microscopy was used to examine biofilms. Results: Out of 25 enterococcal isolates from asymptomatic patients, 9 (36%) isolates were found to be biofilm producers (6 weak [optical densities [OD]595 < 0.2] and three medium [OD595≥0.2 to< 0.5]). Twelve (48%) out of 25 enterococcal isolates from UTI cases, produced biofilms (7 weak, 4 medium, and 1 strong [OD595=0.5]). The esp gene was present in 30 (12 biofilm+, 18 biofilm−) isolates. Seventeen esp positive isolates were from asymptomatic cases whereas 13 were from UTI. However, we found that 100% of medium and strong biofilm producers were esp positive (P < 0.001). On comparing Enterococcus faecalis (n = 10) and E. faecium (n = 40) isolates, 70% of E. faecalis isolates were biofilm producers as compared to only 35% of E. faecium isolates (P > 0.05). The esp positivity was observed more in E. faecium isolates (65%) as compared to 40% in E. faecalis. Vancomycin-sensitive enterococcal and vancomycin-resistant enterococcal isolates and catheter-related and unrelated isolates showed similarity in biofilm production and esp positivity. Conclusion: The esp gene is not compulsorily required to produce biofilm but when present may enhance the biofilm formation. We did not find any correlation between biofilm formation and the ability of the strain to cause symptomatic UTI be associated with catheters or vancomycin resistance.