*2.4. Fungal Starter Cultures*

Overnight cultures of fungi were prepared by using stock SDA plates to inoculate RPMI-1640 without bicarbonate, supplemented with 3-(N-morpholino)propane sulfonic acid (MOPS), 2% (*w*/*v*) glucose, and pH adjusted to 7 with 10 M NaOH. Then, the stocks were incubated statically at 30 ◦C for 24 h. C. albicans and C. auris pseudo-hyphae were produced using conditions published previously [44]. Briefly, overnight cultures of Candida species were collected by centrifugation at 1500 G for 15 min at 4 ◦C, washed twice with 0.15 M NaCl, resuspended in 0.15 M NaCl, and incubated at room temperature for 24 h to induce starvation. After 24 h of starvation, cells were inoculated into RPMI 1640 at a final concentration of 1 × <sup>10</sup><sup>6</sup> cells/mL and incubated at 37 ◦C with shaking for 6 h.

#### *2.5. Production and Purification of Bacterial Cellulose*

BC production was carried out following the protocol reported in our previous paper [36]. Briefly, starter cultures of *G. xylinus* were used to inoculate HS media and incubated statically for 14 days at 37 ◦C. Following the incubation period, bacterial cellulose (BC) pellicles that formed as a raft on the surface of the HS media were aseptically harvested. The unpurified BC was initially heated to 100 ◦C in distilled water, which was followed by the addition of 2% (*w/v*) sodium hydroxide and reheating to 100 ◦C in fresh distilled water for a further hour or until the BC became fully transparent. Then, each pellicle was frozen at −20 ◦C before lyophilisation.

#### *2.6. Preparation of Working Solutions and Loading of Antifungal Agents*

Stock solutions, working solutions, and dilution series' were conducted according to ISO 16256:2012(en) [45]. Briefly, 12,800 mg/L thymoquinone, ocimene, or miramistin and 3200 mg/L amphotericin B were made by dissolving each compound in DMSO. A dilution series following Tables 1 and 2 were conducted to obtain working solutions at 200-fold the final concentration. Then, a 1:100 dilution was conducted to produce drug concentrations at twice the final concentration. The final concentration of amphotericin B ranged from 0.03 to 16 mg/L, and thymoquinone, ocimene, and miramistin ranged from 0.125 to 64 mg/L. Clean lyophilised BC pellicles were aseptically cut into 4 mm (used in cytotoxicity assays to fit 96-well plates) and 8 mm disks using a biopsy punch, which was then submerged in the varying concentrations of either thymoquinone, ocimene, miramistin, or amphotericin B and were placed on an orbital shaker (150 rpm) for 24 h in the dark at room temperature. Confirmation of loading was achieved through FTIR analysis


**Table 1.** Scheme for preparing amphotericin B in DMSO with a final concentration of 0.06 to 16 mg/L.

**Table 2.** Scheme for preparing thymoquinone, ocimene, and miramistin in DMSO 0.26 to 64 mg/L.

