© 2019 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd. Epidemiological studies comparing clinical and commensal Staphylococcus epidermidis isolates suggest that biofilm formation is a discriminant biomarker. A study showed that four non-biofilm-forming clinical S. epidermidis isolates could form an induced biofilm by trypsin treatment, suggesting that S. epidermidis can form biofilms in a protease-independent way and in a trypsin-induced way. In this study, the trypsin capacity to induce biofilm formation was evaluated in non-biofilm-forming S. epidermidis isolates (n = 133) in order to support this mechanism and to establish the importance of total biofilms (meaning the sum of protease-independent biofilm and trypsin-induced biofilm). Staphylococcus epidermidis isolates from ocular infections (OI; n = 24), prosthetic joint infections (PJI; n = 64), and healthy skin (HS-1; n = 100) were screened for protease-independent biofilm formation according to Christensen's method. The result was that there are significant differences (p <.0001) between clinical (43.2%) and commensal (17%) protease-independent biofilm producers. Meanwhile, non-biofilm-forming isolates were treated with trypsin, and biofilm formation was evaluated by the same method. The number of commensal trypsin-induced biofilm producers significantly increased from 17% to 79%. In contrast, clinical isolates increased from 43.2% to 72.7%. The comparison between clinical and commensal total biofilm yielded no significant differences (p =.392). A similar result was found when different isolation sources were compared (OI vs. HS-1 and PJI vs. HS-1). The genotype icaA−/aap+ was associated with the trypsin-induced biofilm phenotype; however, no correlation was observed between aap mRNA expression and the level of trypsin-induced biofilm phenotype. Studying another group of commensal S. epidermidis non-biofilm-forming isolates (HS-2; n = 139) from different body sites, it was found that 70 isolates (60.3%) formed trypsin-induced biofilms. In conclusion, trypsin is capable of inducing biofilm production in non-biofilm-forming commensal S. epidermidis isolates with the icaA−/aap+ genotype, and there is no significant difference in total biofilms when comparing clinical and commensal isolates, suggesting that total biofilms are not a discriminant biomarker.