Macroalgae are a rich source of polyphenols, and their ingestion promotes various health benefits. However, information on factors contributing to health benefits such as antioxidants, antimicrobial properties, bioaccessibility, and cytotoxicity is less explored and often unavailable. Therefore, this study aims to investigate the above-mentioned parameters for the brown and green macroalgae Sargassum wightii and Ulva rigida, respectively, collected from the southeast coast of India. S. wightii exhibited higher antioxidant activity and moderate antimicrobial activity against major food pathogens in an agar well diffusion assay and in the broth microdilution method (MIC50 being <0.5 mg/mL for all microorganisms tested). Both macroalgae extracts exhibited significantly high bioaccessibility of polyphenols. To evaluate the safety of the extracts, in vitro cytotoxicity by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was carried out on the primary cells: mouse splenic lymphocytes. An almost complete decline in the cell viability was seen at considerably high concentration (50 mg/mL), expressing the reasonably high safety of the extracts. The extracts of both macroalgae were quantified for polyphenols, wherein fucoxanthin (9.27 ± 2.28 mg/kg DW) and phloroglucinol (17.96 ± 2.80 mg/kg DW) were found to be greater in the S. wightii apart from other phenolics, like gallic acid, quercetin, vanillin, and ferulic acid. The results signify the tremendous scope for the value addition of S. wightii through extraction and purification of polyphenols for its potential exploitation in functional foods and nutraceuticals or as an antimicrobial ingredient in active or smart packaging.