Phyla nodiflora is a creeping perennial herb, which is widely used in folk medicine. The present study evaluates the phytochemical constituents and in vitro antioxidant and antibacterial potential of the aqueous leaf extract of Phyla nodiflora. The phytochemical analyses were carried out using standard methods. The antioxidant activity of aqueous leaf extract was investigated by using DPPH and total antioxidant activity. The antibacterial assay was determined using the agar well diffusion method against the Gram-positive and Gram-negative bacterial strains such as Staphylococcus aureus, Bacillus subtilis, Klebsiella pneumoniae and Pseudomonas aeruginosa using streptomycin as a positive control. The aqueous leaf extract of Phyla nodiflora was screened against Human Hepatocarcinoma (HepG2) cell lines to determine its anticancer activity. Phytochemical screening of aqueous leaf extract revealed the presence of steroids, flavonoids, carbohydrates, tannins, saponins and alkaloids. Aqueous leaf extract showed DPPH radical scavenging and total antioxidant activity with an IC₅₀ value of 45.91 µg/ml and 52.33 µg/ml.  The highest zone of inhibition was found in K. pneumoniae (22.5mm), while P. aeruginosa (9.1mm) exhibited the lowest zone of inhibition, indicating that the leaf extract has significant antibacterial properties. The aqueous leaf extract of P. nodiflora exhibited significant antiproliferative activity against the HepG2 cancer cell line with an IC₅₀ value of 47.5 μg/ml. The increased apoptotic potential of HepG2 cells in a concentration-dependent manner was determined using the Acridine Orange/Ethidium Bromide assay. It has been concluded that the aqueous leaf extract of Phyla nodiflora possesses the potential for antioxidant, antimicrobial and anticancer activities.