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Novel biodegradable and biocompatible formulations of “old” but “gold” drugs such as nystatin (Nys) and amphotericin B (AmB) were made using a biopolymer as a matrix. Medium chain length polyhydroxyalkanoates (mcl-PHA) were used to formulate both polyenes (Nys and AmB) in the form of films (~50 µm). Thermal properties and stability of the materials were not significantly altered by the incorporation of polyenes in mcl-PHA, but polyene containing materials were more hydrophobic. These formulations were tested in vitro against a panel of pathogenic fungi and for antibiofilm properties. The films containing 0.1 to 2 weight % polyenes showed good activity and sustained polyene release for up to 4 days. A PHA monomer, namely 3-hydroxydecanoic acid (C10-OH), was added to the films to achieve an enhanced synergistic effect with polyenes against fungal growth. Mcl-PHA based polyene formulations showed excellent growth inhibitory activity against both <i>Candida</i> yeasts (<i>C. albicans</i> ATCC 1023, <i>C. albicans</i> SC5314 (ATCC MYA-2876), <i>C. parapsilosis</i> ATCC 22019) and filamentous fungi (<i>Aspergillus fumigatus</i> ATCC 13073; <i>Trichophyton mentagrophytes</i> ATCC 9533, <i>Microsporum gypseum</i> ATCC 24102). All antifungal PHA film preparations prevented the formation of a <i>C. albicans</i> biofilm, while they were not efficient in eradication of mature biofilms, rendering them suitable for the transdermal application or as coatings of implants.