The gut microbiota plays a central role in human health, contributing to immune regulation, metabolic function, and protection against enteric pathogens, with fermentable dietary substrates acting as key modulators of its composition and activity. Milk has long been a dietary staple valued for its calcium, protein, and vitamin D contents and established role in bone health. Therefore, cow's milk and dairy products feature prominently in food-based dietary guidelines (1). However, growing consumer interest in environmental sustainability, farm animal welfare, and perceived health benefits has driven a shift toward plant-based dairy milk alternatives (PBDMA) in Australia, with an estimated 30% increase in PBDMA consumption between 2019 and 2021 (2). Alongside this dietary shift, global demand for smoothies and functional fruit-based beverages continues to grow, driven by consumer interest in convenient, nutrient-rich options that align with health-conscious lifestyles. Yet the comparative impact of dairy versus plant-based dairy milk alternative (PBDMA) matrices on gut microbiota modulation, particularly when combined with fruit-derived bioactive compounds such as those in strawberries, remains poorly understood. This study therefore examined full cream dairy milk (selected from 3 commercial options) and regular soy milk (selected from 9 commercial PBDMAs based on sensory acceptability and probiotic survivability), combined with strawberry as smoothies (strawberry blended with dairy milk, soy milk, or water), on in vitro gut microbiota modulation. Treatments (strawberry-dairy, SD; strawberry-soy, SS; strawberry-water, SW; water-dairy, WD; water-soy, WS) underwent simulated in vitro gastrointestinal digestion followed by colonic fermentation using human faecal inoculum. Gut microbial composition was assessed via 16S rRNA sequencing at 0 h (inoculum), 12 h, and 24 h, relative to an unfermented control. At the phylum level, Bacillota, Bacteroidota, and Actinomycetota dominated all samples (>85% combined relative abundance). Bacillota and Bacteroidota are major short-chain fatty acid producers supporting gut barrier integrity and metabolic health, while Actinomycetota contribute to immune modulation (3). Bacillota was highest in SS (59.1% at 24 h), whereas Bacteroidota was highest overall in WD at 12 h (36.2%) and, among strawberry smoothies, in SD at 24 h (28.0%). Actinomycetota peaked in control and WS at 12 h (26.6% and 28.3%). However, statistical evaluation (n = 4/treatment) showed a significant difference (p < 0.05) for Bacteroidota but not Bacillota or Actinomycetota (p > 0.05). These findings demonstrate that dairy milk and strawberry matrices differentially affect the dominant gut bacterial phyla during colon fermentation, with strawberry-dairy combinations significantly better maintaining Bacteroidota populations and strawberry-soy combinations showing numerically higher, though not statistically significant, Bacillota abundance. However, in vivo human studies are warranted to confirm these in vitro findings and evaluate their relevance to gut microbial composition and function in humans.