As part of the chemical investigation into some medicinal plants growing in Tunisia and in order to continue the search for potential biological natural compounds, two new alkaloid derivatives of the 3-benzazocine skeleton (cotuzine A and B) and a new polyphenolic substance (3-(1,3-dihydroxyprop-2-yl)-4-hydroxyanisol, corimen) were isolated from the aerial part (stems, leaves) of Cotula coronopifolia (L.). The structures of these three new compounds were established by spectroscopic procedures (1H, 13C, one- and two-dimensional NMR). The stereochemistry of the new alkaloids was determined based on different NOE effects revealed on the NOESY spectrum, and the structures were confirmed by mass spectrometry and IR spectroscopy. Compound 1 showed strong activity against Citrobacter freundii (inhibitory zone IZ = 9.5 mm; minimal inhibition concentration MIC = 500 g/mL) and Pseudomonas aeruginosa (IZ = 8.5 mm; MIC = 500 g/mL), as well as antifungal activity toward Trichophyton rubrum (54%) and Aspergillus fumigatus (58%).