An increase in antifungal resistance has seen a surge in fungal wound infections in patients who are immunocompromised resulting from chemotherapy, disease, and burns. Human pathogenic fungi are increasingly becoming resistant to a sparse repertoire of existing antifungal drugs, which has given rise to the need to develop novel treatments for potentially lethal infections. Bacterial cellulose (BC) produced by Gluconacetobacter xylinus has been shown to possess many properties that make it innately useful as a next-generation biopolymer to be utilised as a wound dressing. The current study demonstrates the creation of a pharmacologically active wound dressing by loading antifungal agents into a biopolymer hydrogel to produce a novel wound dressing. Amphotericin B is known to be highly hepatotoxic, which reduces its appeal as an antifungal drug, especially in patients who are immunocompromised. This, coupled with an increase in antifungal resistance, has seen a surge in fungal wound infections in patients who are immunodeficient due to chemotherapy, disease, or injury. Antifungal activity was conducted via Clinical & Laboratory Standards Institute (CLSI) M27, M38, M44, and M51 against Candida auris, Candida albicans, Aspergillus fumigatus, and Aspergillus niger. This study showed that thymoquinone has a comparable antifungal activity to amphotericin B with mean zones of inhibition of 21.425 ± 0.925 mm and 22.53 ± 0.969 mm, respectively. However, the mean survival rate of HEp-2 cells when treated with 50 mg/L amphotericin B was 29.25 ± 0.854% compared to 71.25 ± 1.797% when treated with 50 mg/L thymoquinone. Following cytotoxicity assays against HEp-2 cells, thymoquinone showed a 71.25 ± 3.594% cell survival, whereas amphotericin B had a mean cell survival rate of 29.25 ± 1.708%. The purpose of this study was to compare the efficacy of thymoquinone, ocimene, and miramistin against amphotericin B in the application of novel antifungal dressings.