Abstract The antimicrobial activity of an essential Roman chamomile flower oil from the Provence (France) was tested against various strains of Gram-positive bacteria (Staphylococcus aureus and Enterococcus faecalis) and Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa, Proteus vulgaris, Klebsiella pneumoniae and Salmonella sp.) as well as against the yeast Candida albicans using a modified agar dilution and agar diffusion method. In addition, some pure main and minor compounds (chemical composition obtained by means of GC and GC/MS measurements), such as isobutyl angelate (32.1%), 2-methylbutyl angelate (16.2%), isobutyl isobutyrate (5.3%), methyl 2-methylbutyrate (1.9%), prenyl acetate (1.4%), 2-methylbutyl 2-methylbutyrate (1.2%) and 2-methylbutyl acetate (1.2%), were also studied for their antimicrobial effects. The Roman chamomile sample showed high antimicrobial activity against all strains of tested microbes (reference compounds: eugenol and three synthetic antibiotics). A similar result was found for 2-methylbutyl 2-methylbutyrate, 2-methylbutyl acetate and prenyl acetate. Surprisingly, no antimicrobial effects against Escherichia coli by methyl 2-methylbutyrate and against Klebsiella pneumoniae by 2-methylbutyl angelate and methyl 2-methylbutyrate as well as by isobutyl isobutyrate against Staphylococcus aureus were observed. It is proposed that the very high antimicrobial activity of Roman chamomile oil from the Provence results of effects found for both main and minor constituents of this oil.