Purpose <p>This study aimed to optimize the fermentation of waste yarrow (<i>Achillea millefolium</i> L.) powder using <i>Lacticaseibacillus rhamnosus</i> A71 and <i>Saccharomyces boulardii</i> to enhance total polyphenol content (TPC) and improve key biological activities, including antioxidant, antimicrobial, and acetylcholinesterase inhibitory effects.</p> Methods <p>Fermentation was conducted using <i>L. rhamnosus</i> A71 and <i>S. boulardii</i>, with response surface methodology employed to determine optimal fermentation parameters: fermentation time, inoculum concentration, nutrient broth concentration, and liquid-to-solid ratio. The extracts were analyzed for TPC, antioxidant activity (radical scavenging and reducing power), antimicrobial activity, acetylcholinesterase inhibition, and polyphenolic profile using HPLC.</p> Results <p>A single day of fermentation with <i>L. rhamnosus</i> A71 increased TPC by 44%, enhanced radical scavenging activity by 11%, and improved reducing power by 42%. Two-day fermentation with <i>S.&#xa0;boulardii</i> led to a 25% increase in TPC with moderate enhancements in antioxidant capacity. Antimicrobial activity was significantly enhanced, notably with <i>Staphylococcus aureus</i> MIC values reduced up to 16-fold, while acetylcholinesterase inhibition was enhanced, indicating an overall improvement in the functional bioactivity of the extract. Fermentation altered the extract’s phytochemical profile: apigenin 7-O-glucoside remained dominant in the unfermented and <i>S.&#xa0;boulardii-</i>fermented samples, whereas quercetin became the most abundant compound following fermentation with <i>L. rhamnosus</i> A71. Levels of caffeic acid, cyanidin chloride, and quercetin increased across both fermented extracts, while rutin content decreased.</p> Conclusion <p>Microbial fermentation substantially enhanced the bioactive potential of yarrow waste extracts, improving polyphenol yield and modifying phytochemical composition in a functionally beneficial way. These findings highlight the potential of fermentation as a green biotechnological tool for converting plant-based waste materials into high-value ingredients suitable for use in nutraceuticals, functional foods, and pharmaceutical applications.</p> Statement of Novelty <p>This study introduces a sustainable way to turn waste material from yarrow herb processing into valuable extracts rich in natural antioxidants. By using safe, food-grade microbes to ferment the plant residue, we were able to increase the concentration and biological activity of polyphenols—compounds known for their health benefits. We also used microwave energy to extract these compounds more efficiently. While yarrow has been studied before, this is the first time its industrial waste has been improved through fermentation and green extraction. This research shows a new way to reduce plant-based waste and produce bioactive ingredients that could be used in food, supplements, or related applications.</p> Graphical Abstract <p></p>