Anthemis species are traditionally used to treat infectious and inflammatory processes, among others clinical disturbances. In the current study, the chemical composition, the total phenolic and flavonoid contents, the antioxidant, anticholinesterase, antimicrobial, and antibiofilm activities of Anthemis stiparum subsp. sabulicola aerial parts methanolic extract (As-ME) and essential oil (As-EO) were investigated. The chemical composition of As-EO was established by GC-MS and GC-FID. Total phenolic and flavonoid contents of As-ME were spectrophotometrically determined. Diphenyl-1-picrylhydrazyl (DPPH center dot) radical scavenging, cupric reducing antioxidant capacity (CUPRAC) and beta-carotene bleaching assays were applied to evaluate the antioxidant potential. The anticholinesterase activity against acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) enzymes were carried out spectrophotometrically. The antimicrobial activity was assessed by Minimal Inhibitory Concentration (MIC) using broth microdilution method against 7 ATCC(center dot) bacterial and one ATCC(center dot) yeast reference strains. The antibiofilm effect was determined quantifying the percentage of adhesion inhibition. GC-MS and GC-FID identified 72 compounds (99.02%), being As-EO predominantly constituted by germacrene D (11.13%), t-cadinol (11.01%), camphor (6.73%), spathulenol (6.50%) and isoamyl salicylate (6.45%). The total phenolic and flavonoid contents of As-ME were 13.6 +/- 0.03 and 5.9 +/- 0.04 pyrocatechol equivalents and quercetin equivalents, respectively. In beta-carotene-linoleic acid assay, As-ME showed the best lipid peroxidation inhibition activity with an IC50 = 9.96 mu g/mL followed by As-EO with an IC50 = 619.98 mu g/mL. In contrast, in DPPH assay, As-ME and As-EO showed moderate to low activity with an IC50 = 92.69 mu g/mL for As-ME and 917.69 mu g/mL for As-EO. While in CUPRAC assay, As-EO and As-ME indicated a less to moderate reducing activity. As-ME inhibited AChE (IC50 = 490.46 mu g/mL) and BChE (IC50 = 142.07 mu g/mL), while As-EO was inactive against AChE and revealed a discreet inhibitory action against BChE (IC50 = 212.14 mu g/mL). As-ME displayed better antimicrobial activity than As-EO, being active against Staphylococcus aureus (ATCC(center dot) 25923) and Bacillus subtilis (ATCC(center dot) 6633), with MIC of 1.56 mg/mL. An expressive fungal adhesion inhibition (80.02%) on Candida albicans (ATCC(center dot) 10239) was detected with As-ME at 6.25 mg/mL. These results showed that A. stiparum subsp. sabulicola is a natural source of active compounds with antibiotic and antibiofilm effects against S. aureus and B. subtilis, and C. albicans, respectively, and also presents antioxidant and anticholinesterase properties.