Populus ciliata Wall. ex Royle (Salicaceae family) is a fast-growing, multipurpose native tree of the Himalayas. Traditionally, the tree's bark is used for blood purification, treatment of rheumatism, and fatigue, and to relieve the pain of menstrual cramps. The present study deals with screening crude methanolic extracts and fractions (n-hexane, chloroform, ethyl acetate, and n-butanol) of P. ciliata bark and leaves for total polyphenolic contents, antioxidant, antimicrobial, anti-inflammatory, and cytotoxic activities. Results showed that among all tested samples, the bark ethyl acetate fraction had the highest polyphenolic contents and strong antioxidant potential (DPPH 25.75±3.60 µg/mL; ABTS 33.55±2.96 µg/mL; FRAP 70.39±1.75 µM of Fe equivalents). The HPTLC analysis confirmed the presence of gallic acid, salicin, and quercetin molecules in the extracts and different fractions of P. ciliata whereas ethyl acetate fractions showed their maximum quantity. The ethyl acetate fraction of bark also revealed higher antibacterial activity against Pseudomonas aeruginosa (MIC 0.0156 mg/mL), Klebsiella pneumoniae and Staphylococcus aureus (MIC 0.0312 mg/mL), Bacillus subtilis and Escherichia coli (MIC 0.0625 mg/mL), and antifungal activity against Fusarium oxysporum (MIC 0.125 mg/mL) and Rosellinia necatrix (0.250 mg/mL), whereas butanol fraction of bark showed maximum antifungal activity against Candida auris (MIC 1.25 mg/mL). The highest anti-inflammatory activity was observed in bark ethyl acetate fraction (IC50 0.113±0.017 mg/mL for egg albumin and 0.160±0.014 mg/mL for bovine serum albumin assay) in comparison with other extracts and fractions. Additionally, bark crude extract and its ethyl acetate fraction were observed more effective against colon cancer cell lines (IC50 values <0.170 mg/mL for HCT-116 and <0.340 mg/mL for SW-620). The study concludes that the bark ethyl acetate fraction is the most biologically active fraction due to a higher number of polyphenolic contents (salicin, gallic acid, and quercetin) and among both plant parts, bark has a higher medicinal value than leaves. Therefore, this fraction can be further explored to extract biologically active compounds of pharmaceutical, food, and cosmetic values.