The members of the Liliaceae family are considered an excellent source of biologically active compounds. However, work on antimicrobial potential and characterization of the bioactive fractions of the Lilium philadelphicum flower is limited and needs to be explored. The present study reports the antimicrobial potential of the bioactive fraction extracted from the flower of L. philadelphicum (red lily) and partial characterization of the bioactive compound(s). The antimicrobial activity was tested against nine different Gram-positive and Gram-negative bacterial strains. The minimum inhibitory concentration (MIC) values of methanolic extract of the L. philadelphicum flower against Acinetobacter bouvetii, Achromobacter xylosoxidans, Bacillus subtilis MTCC 121, Candida albicans MTCC 183, Klebsiella pneumoniae MTCC 3384, and Salmonella typhi MTCC 537 were 25, 50, 12.5, 50, 100, and 50 μg/mL, respectively. The phytochemical analysis of the extract revealed the presence of phenols, flavonoids, tannins, terpenoids, glycosides, coumarins, and quinones. The cytotoxicity of the partially purified compound against the HepG2 cell line using MTT assay demonstrated up to 90% cell viability with a bioactive compound concentration of 50 μg/mL. However, the increase in the bioactive compound’s concentration up to 1000 μg/mL resulted in nearly 80% cell viability. This minor decline in cell viability suggests the importance and suitability of the bioactive compound for therapeutic applications. Spectroscopic studies of the bioactive compound by UV-visible spectroscopy, FT-infrared spectroscopy, gas chromatography-mass spectrometry (GC-MS), as well as phytochemical analysis, suggested the presence of a terpenoid moiety, which may be responsible for the antimicrobial property of the L. philadelphicum flower.