Marine invertebrates represent a valuable reservoir of pharmaceutical bioactive compounds with potential relevance to various medical applications. These compounds exhibit notable advantages when compared to their terrestrial counterparts, in terms of their potency, activity, and mechanism of action. Within this context, the present work aimed to extract, chemically characterize, and investigate the bioactivity of the gonadal extract of the sea urchin Paracentrotus lividus (P. lividus) collected along the Mediterranean coast of Alexandria, Egypt. Fractions of the gonadal extract were characterized by Spectrophotometry and gas chromatography-mass spectrometry (GC-MS), and their bioactivities were investigated in vitro. The analysis supported the extract richness of carotenoids and bioactive compounds. The extract showed promising anticancer activity against three different breast cancer cell lines with different levels of aggressiveness and causative factors, namely MDA-MB-231, MDA-MB-453, and HCC-1954. Gene expression analysis using RT-qPCR showed that P. lividus extract inhibited the expression of crucial factors involved in cell cycle regulation and apoptosis. In addition, the extract significantly inhibited the lipo-polysaccharides (LPS) induced inflammation in the RAW264.7 macrophage cell line and exerted anti-bacterial activity against the Gram-negative bacteria Klebsiella pneumoniae and Pseudomonas aeruginosa. Collectively, these results demonstrated the chemical richness and the wide-scale applicability of P. lividus gonadal extract as an anti-cancer, anti-bacterial, and anti-inflammatory natural extract.