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Molecular mechanisms of biofilm formation in <i>Candida tropicalis</i> and current methods for biofilm analyses in this fungal pathogen are limited. (2) Methods: Biofilm biomass and crystal violet staining of the wild-type and each gene mutant strain of <i>C. tropicalis</i> were evaluated on silicone under synthetic urine culture conditions. (3) Results: Seven media were tested to compare the effects on biofilm growth with or without silicone. Results showed that biofilm cells of <i>C. tropicalis</i> were unable to form firm biofilms on the bottom of 12-well polystyrene plates. However, on a silicone-based platform, Roswell Park Memorial Institute 1640 (RPMI 1640), yeast nitrogen base (YNB) + 1% glucose, and synthetic urine media were able to induce strong biofilm growth. In particular, replacement of Spider medium with synthetic urine in the adherence step and the developmental stage is necessary to gain remarkably increased biofilms. Interestingly, unlike <i>Candida albicans</i>, the <i>C. tropicalis</i> <i>ROB1</i> deletion strain but not the other five biofilm-associated mutants did not cause a significant reduction in biofilm formation, suggesting that the biofilm regulatory circuits of the two species are divergent. (4) Conclusions: This system for <i>C. tropicalis</i> biofilm analyses will become a useful tool to unveil the biofilm regulatory network in <i>C. tropicalis</i>.