The present research work lays great emphasis on phytochemical investigation and biological screening of a medicinal plant Ruellia squarrosa belonging to the family Acanthaceae. Dichloromethane and methanol extracts of the aerial parts and roots of Ruellia squarrosa were prepared. The preliminary phytochemical investigation of selected plant indicated the presence of anthraquinone glycosides, cardiac glycosides, flavonoids, alkaloids and saponins while tannin was absent. The root and aerial extract of the plant in both solvents were subjected to biological screening such as immunomodulatory inhibitory activity, antiglycation activity, carbonic anhydrase inhibitory activity, xanthine oxidase inhibitory activity, α-chymotrypsin inhibitory activity and tyrosine inhibitory activity. Dichloromethane and methanol extracts of aerial parts and roots of Ruellia squarrosa were also examined for anticancer activity, antifungal activity, antibacterial activity, brine-shrimp cytotoxic activity, antioxidant activity and antileishmanial activity. The dichloromethane extract of aerial parts of the plant showed significant immunomodulatory inhibitiory activity with IC50 39.48±8.06 compared with other extracts using ibuprofen as standard. The dichloromethane extract of aerial parts of the plant showed significant antiglycation effect with IC50 382.21±3.43 using rutin as standard drug as among other extracts. The methanol extract of the aerial parts of the plant showed significant urease inhibitory activity with IC50 130.2±0.57 using thiourea as standard drug. The methanol extract of the aerial parts of the plant showed significant activity against a carbonic anhydrase inhibitory activity with IC50 1656.7±0.08 using acetazolamide as standard as among other extracts. The dichloromethane extract of root parts of the plant showed significant tyrosine inhibitiory activity with IC50 32.2±0.21 compared with other extracts using Kojic acid as standard. The dichloromethane extract of root parts of the plant showed significant α-chymotrypsin inhibitory activity using chymostatin as standard drug as among other extracts. The dichloromethane and methanol extracts of roots and aerial parts of Ruellia squarrosa were evaluated for anticancer activity by MTT assay method against human prostate cancer (PC3) cell line. The dichloromethane extract of roots of the plant showed significant anticancer activity against PC3 cells line with IC50 15.4 ± 0.3 using doxorubicin as standard drug. The dichloromethane and methanol extracts of roots and aerial parts of Ruellia squarrosa were evaluated for brine shrimp lethality activity. The dichloromethane extract of aerial parts and roots of plant showed significant activity having 93% mortality at concentration of 1000 μg /ml as among other extracts. The antifungal activity of plant extracts was carried out against different fungal strains named as Tricinshophyton longifusis, Candida albicans, Candida glabarata, Fusarium solani, Microsporum canis, and Aspergillus flavus. The dichloromethane and methanol extracts of aerial parts and roots of the plant showed no significant antifungal activity. The antibacterial activity of plant extract were determined against the five different strains of bacteria, two of them were Gram negative bacteria (Escherichia coli, Salmonella typhi) and three were Gram positive bacteria (Bacillus spizizenii, Staphylococcus aureus, Staphylococcus epidermidis). The dichloromethane and methanol aerial parts and roots of the plant extracts showed no significant antibacterial activity. The dichloromethane and methanol extracts of roots and aerial parts of Ruellia squarrosa were evaluated for antioxidant activity, xanthine oxidase inhibitory activity and antileishmanial activity. The results showed no significant activity. The dichloromethane and methanol extracts of the roots and aerial parts of Ruellia squarrosa were subjected to phytochemical investigation and nine novel compounds were isolated. Four novel compounds were obtained from methanolic extracts and five novel compounds from dichloromethane extract of the plant Ruellia squarrosa.The structures of isolated novel compounds have been elucidated on the basis of spectral data like UV, IR, 1H-NMR, 13C-NMR and HR-EIMS spectra. The compounds have been named on the basis of species under study Squarrosal, Squarrosol, Squarroic acid, Squarros, Squarrasadole, Squarostriol, Squarrapyranoic acid, Squarrosone and Squarrosian.The four compounds Squarroic acid, Squarrosone, Squarrasadole and Squarrapyronic acid belongs to the glycoside class. Two compounds belongs the flavonoids class such as Squarrosal and Squarrosol. One compound Squarros belong to natural phenolic compound class. Remaining two compounds belongs to the steroidal class named as Squarostriol and Squarrosian.The novel compounds would be helpful in the treatment of cancer and other ailments, drug designing and development.