Globularia alypumL. (GA) is a Mediterranean plant of theGlobulariaceaefamily which is widely used in traditional Tunisian medicine. The main goal of this study was to evaluate the phytochemical composition, antioxidant, antibacterial, and antibiofilm activities, and the antiproliferative potential of different extracts of this plant. The identification and the quantification of the different constituents of extracts were determined using gas chromatography–mass spectrometry (GC-MS). The antioxidant activities were evaluated using spectrophotometric methods and chemical tests. The antiproliferative study was based on the use of colorectal cancer SW620 cells, including an antibacterial assessment with the microdilution method and analysis of the antibiofilm effects via the crystal violet assay. All extracts presented several components, mainly sesquiterpenes, hydrocarbon, and oxygenated monoterpenes. The results revealed that the maceration extract had the most important antioxidant effect (IC50= 0.04 and 0.15 mg/mL), followed by the sonication extract (IC50= 0.18 and 0.28 mg/mL). However, the sonication extract demonstrated significant antiproliferative (IC50= 20 µg/mL), antibacterial (MIC = 6.25 mg/mLand MBC > 25 mg/mL), and antibiofilm (35.78% at 25 mg/mL) properties againstS. aureus. The results achieved confirm the important role of this plant as a source of therapeutic activities.