Prebiotics are dietary components that resist digestion in the upper gastrointestinal tract and promote the growth and functional activity of beneficial microorganisms in the gut, thereby contributing to intestinal health and overall well-being (Roshani et al., 2026). Dragon fruit (Selenicereus undatus) is a nutrient-rich fruit containing dietary fibre and bioactive compounds, while its peel, which is commonly considered a by-product, may represent a valuable source of functional food ingredients. This study investigated the prebiotic potential of S. undatus peel and pulp using Lactobacillus acidophilus cultured in freshly prepared MRS (De Man, Rogosa and Sharpe) broth. L. acidophilus grown in MRS broth without peel or pulp extract was considered the control. The effects of peel and pulp on probiotic-related functional properties were evaluated based on auto-aggregation, coaggregation with Escherichia coli, cell surface hydrophobicity and autolysis. Auto-aggregation increased progressively from 1 to 5 h in all treatments, with the highest values observed in peel-treated cells, increasing from 44.85 ± 0.42% to 78.65 ± 0.38%, followed by pulp-treated cells, which increased from 31.24 ± 0.28% to 61.82 ± 0.31%, and control cells, which increased from 23.15 ± 0.30% to 58.42 ± 0.43%. Coaggregation with E. coli was enhanced in the presence of peel, reaching 60.84 ± 0.21%, compared with 48.72 ± 0.20% for pulp and 41.35 ± 0.03% for the control, indicating improved interaction between the probiotic cells and the test organism. Cell surface hydrophobicity was also higher in peel-treated cells (58.92 ± 0.46%), followed by pulp-treated cells (45.76 ± 0.38%) and control cells (41.85 ± 0.42%), suggesting enhanced cell surface characteristics associated with microbial adhesion. In contrast, autolysis was lower in peel-treated cells (18.42 ± 0.35%) than in pulp-treated cells (22.68 ± 0.30%) and control cells (25.15 ± 0.38%), suggesting greater cellular stability and reduced self-degradation. Data were analysed using one-way analysis of variance (ANOVA) followed by the least significant difference (LSD) test, with significance set at p < 0.05. The comparatively higher values observed across the functional characteristics of L. acidophilus in the peel treatment indicate that the peel may provide favourable substrates or compounds that support probiotic performance. In addition, the improved aggregation and hydrophobicity may contribute to the ability of probiotic cells to interact with intestinal surfaces, while enhanced coaggregation may be beneficial for competitive interactions with potentially harmful microorganisms. Overall, the findings demonstrate that S. undatus peel exhibited greater prebiotic potential than pulp, as evidenced by its favourable effects on aggregation, coaggregation, cell surface hydrophobicity and cellular stability of L. acidophilus. These findings highlight the potential of S. undatus peel as a functional ingredient for the development of probiotic and prebiotic food products and support the sustainable utilisation of dragon fruit processing by-products (Villafán-González et al., 2025).