The objective of this study was to investigate the antioxidant potential, antimicrobial, haemolytic and cytotoxic activities of petroleum ether extract of Padina gymnospora. The total phenolic and flavonoid content was estimated by Folin’s ciocalteau and Aluminium chloride methods. Antioxidant potential of the extract was estimated by Phosphomolybdneum method, Diphenyl picryl hydrazine free radical scavenging method and the Superoxide free radical methods. Antimicrobial activity was performed by Disc diffusion method against seven bacterial and five fungal strains. P.gymnospora exhibited 61.52 mg GAE/g equivalents of phenolic content, 54.2 ± 0.15 mg Quercetin/grams equivalents of flavonoid content. The total antioxidant activity was found to be 74.23% and scavenging activity of DPPH 62.9% and superoxide ion 59.4% at 1000µg/ml. P. gymnospora extract has shown strong activity against E. coli, S. aureus, A. niger, C. albicans and moderate activity against B. cereus, B. subtilis, K. pneumoniae, P. aeroginosa, A. flavus, A fumigatus, T. viridae and T. rubrum. The crude extract was screened for the haemolytic activity towards human erythrocytes. The haemolytic activity was performed by spectroscopic method for different concentrations (250, 500, 750, and 1000) µg/ml) and it has shown the dose dependent hemolysis activity. The cytotoxic activity of crude extract against Hel 92.1.7 leukaemia cell line was evaluated by MTT assay which shown remarkable decrease in the number of cells by dose dependent manner with IC₅₀ of 102.3 µg /ml. The cytotoxic activity was found might be due to the induction of apoptosis in Hel 92.1.7 leukaemia cell line which was demonstrated by DNA fragmentation analysis. The present findings showed that the petroleum ether extracts of Padina gymnospora have strong antioxidant activity, antimicrobial, haemolytic as well as cytotoxic activity which might be due to the presence of phenolic compounds and other secondary metabolites.