The leatherleaf slug Eleutherocaulis alte from Assiut Governorate, Egypt, was investigated using an integrative approach combining morphological, molecular, histological, and bioactivity analyses. Morphological characterization revealed a dorsoventrally flattened body with a brown dorsal surface marked by a pale median line, dark spots, and a narrow central foot. Mitochondrial cytochrome c oxidase I (COI) gene sequencing confirmed its identity as E. alte, showing 98.23% similarity to Laevicaulis alte, and the sequence was deposited in GenBank (OR162029). Scanning electron microscopy demonstrated porous mucus-secreting surfaces essential for locomotion and adhesion, while histological examination revealed distinct secretory cell types within the epidermal and subepidermal layers, including a suprapedal gland producing mixed acidic and neutral mucopolysaccharides. Bioactivity assays indicated that the crude mucus exhibited potent antimicrobial activity, particularly against Bacillus subtilis and Candida albicans, with minimum inhibitory concentrations of 7.8 µg/mL and 3.9 µg/mL, respectively. The crude mucus showed significantly greater antimicrobial activity against Gram-positive and Gram-negative bacteria as well as C. albicans compared to the corresponding positive controls (gentamicin or fluconazole; P < 0.05 − 0.001). However, it exhibited no inhibitory effect against Aspergillus niger, collectively, these findings provide novel taxonomic, anatomical, and biomedical insights into E. alte, and highlight its mucus as a promising natural source of antimicrobial agents.