This study aims to analyze the phytochemical and antimicrobial-related assays including antibacterial, antifungal, antipellicle, and antibiofilm potential of the root of an alpine medicinal plant (Arnebia euchroma (Royle) I.M.Johnst). Ethanolic, methyl acetate, chloroform, and aqueous extracts of root parts of the A. euchroma plant were prepared. The active metabolites of the root part of the plant were evaluated both qualitatively and quantitatively by GC-MS and HPLC techniques, using ethanolic extract. The antimicrobial potential of A. euchroma was investigated using crude extract, fractions, and AgNPs of the A. euchroma root against two Gram-positive bacteria (Enterococcus, Staphylococcus aureus) four Gram-negative bacteria (Salmonella typhi, Pseudomonas aeruginosa, Escherichia coli, Proteus vulgaris) and five fungal strains (Rhizoctonia, Cuneate fasciculus, Aspergillus niger, Fusarium oxysporum, and Candida auris). All the bacterial strains showed sensitivity toward all the fractions (ethanolic crude, chloroform, aqueous, and methyl acetate) and AgNPs. While S. typhi was found the most susceptible, P. vulgaris was most resistant to all extracts and AgNPs. The case of anti-biofilm potential, the S. typhi and Enterococcus were shown to be most susceptible, these bacteria formed biofilm which was disrupted completely by the extracts. Among the fungal strains F. oxysporum and A. niger were the most susceptible and C. fasciculus was the most resistant fungi. All extracts, including ethanolic extract, chloroform, aqueous, and methyl acetate, at concentrations of 12.5 μg/mL and 16 μg/mL and AgNPs showed anti-pellicle activity against S. typhi, Rhizoctonia, and A. niger. Dose-dependent trend in all bioassays was noted. The ethanolic crude and chloroform extracts were the most active followed by methyl acetate and then aqueous extracts. The phytochemical analysis revealed that carbohydrates, alkaloids, saponins, phenols, tannins, proteins, and flavonoids were found in the plant while terpenoids were absent. The present study highlights that root of the A. euchroma is rich in various types of bioactive phytochemicals, which may be responsible for anti-microbial activities toward the tested pathogenic of bacterial and fungal strains. Our results suggest and confirm A. euchroma extracts could be used economically as a valuable bioproduct with new functional properties in the further drug development and pharmaceutical industries.