Abstract <p><i>Candida tropicalis</i>, the most prevalent non-Candida albicans Candida species, is an emerging pathogen forming robust biofilms on medical devices, contributing to biofouling, virulence, and antifungal resistance. In this study, growth conditions for six <i>C. tropicalis</i> clinical isolates (C4, U873, U951, U1179, U1309, U1360) and a standard strain (MTCC-184) were optimized on polypropylene using central composite design-based response surface methodology. The parameters tested included temperature, pH, shaker speed, inoculum size, and incubation time, with biofilm formation quantified by crystal violet, cell viability by MTT, biomass by calcofluor white, and wet/dry weight measurements. Notably, <i>C. tropicalis</i> forms biofilm on polypropylene surfaces, resembling extracellular polymeric substance-rich matrices. Among the isolates, C4, U873, U951, and U1179 fit the CCD model, whereas for MTCC-184, U1309, and U1360, the Johnson Transformation was required to obtain unified optimal conditions. Temperature and pH were the major factors influencing biofilm formation in C4 and U1179, while temperature and incubation time were significant for U873 and U951. A direct correlation was observed between cell viability and biofilm formation, though biomass varied, indicating strain-specific virulence. This high-throughput optimization strategy establishes a platform for antifungal screening, biofilm–material interaction studies, and the development of medical devices resistant to fungal colonization.</p> Key points <p>• <i>Optimized growth conditions of Candida tropicalis biofilm on polypropylene material by RSM</i></p> <p>•&#xa0;<i>Four C. tropicalis isolates fit the CCD model; the other three isolates were modelled using CCD–JT</i></p> <p>•&#xa0;<i>A direct correlation was observed between cell viability and biofilm with variations in cell mass. </i></p> Graphical Abstract <p></p>