Background and Aims Viola tricolor L. (heartsease, wild pansy) is a valuable medicinal plant obtained for pharmaceutical purposes by cultivation. Given that the species is usually strongly colonized by arbuscular mycorrhizal fungi (AMF), we tested in a pot experiment whether these microorganisms were able to influence V. tricolor mass, vitality, and the concentrations of selected elements, phenolic acids and flavonoids. Methods The following treatments were prepared: (1) control: sterile soil without AMF inoculation; (2) Rhizophagus irregularis BEG144; (3) Funneliformis mosseae BEG12; and (4) both isolates. Using a combination of physiological, phytochemical and biophysical methods, we evaluated the effects of these AMF on the performance of heartsease. Results The intensity of mycorrhizal colonization and arbuscule formation was higher when F. mosseae and R. irregularis were introduced separately than when both isolates were present. None of the AMF treatments had an impact either on V. tricolor vitality as expressed by photosynthetic performance index (PI) or on its shoot and root mass. However, in general, a negative correlation was found between the extent of mycorrhizal colonization and shoot mass. We found AMF species specificities in their influence on element, phenolic acid and flavonoid concentrations. Viola tricolor showed no response to F. mosseae. The plants inoculated with R. irregularis had higher concentrations of P, Zn, Mg, and Ca, as well as p-hydroxybenzoic acid and rutin, in comparison to control. Dual AMF species inoculation increased concentrations of Cu, Mg and rutin. Conclusions The enhanced production of secondary metabolites in V. tricolor shoots may be due to improved mineral nutrition by AMF and/or a result of general plant defense reaction to fungal colonization. The tendency towards biomass decrease in AMF treatments could be explained by the allocation of plant carbon both to the maintenance of symbionts and enhanced production of secondary compounds.