It was important to elucidate the mutagenic potential of C. albicans biofilms and the expression of genes related to ACH and ethanol. Mutagenic levels of ACH were produced by control biofilms during incubation in D-glucose at neutral pH. No ACH was produced by biofilms exposed to HICA during D-glucose incubation at acidic pH. At neutral pH, the ACH levels were significantly lower compared to control conditions and below the mutagenic level. At neutral pH, ACH production from ethanol or Dglucose by mature biofilms was decreased similarly by both HICA and caspofungin. Interestingly, although caspofungin inhibited ACH production by early biofilms more than HICA, it resulted in the highest ACH levels at acidic pH during D-glucose incubation. This is relevant as a normal western diet is often rich in D-glucose and the 100 mM concentration used in this study is equivalent to 18 g/l found commonly in food and beverages. In the presence of ethanol, ACH levels produced by all biofilms exposed to HICA or caspofungin were generally lower than in control biofilms. The ethanol concentration of 11 mM used in this study is found in saliva after drinking 0.5 g of alcohol per kg body weight, which is equivalent to 3 glasses of wine for an 80 kg male and thus can be considered clinically relevant. Our results on biofilms support earlier studies on planktonic cell cultures. Biofilms exposed to RPMI and ethanol produced high levels of ACH from ethanol, well above the mutagenic level, and approaching the results obtained with planktonic cultures. It is relevant to point out that the highest ACH levels were produced at later stages of biofilm growth, as would be established in niches of the human body. HICA was most active against C. albicans biofilms at acidic pH, in line with previous studies where planktonic cultures were used. The main focus of the present study was to investigate the anti-biofilm efficacy of HICA against various comparators whereby it was critical to grow the biofilms under standardised conditions before treatment at acidic and neutral conditions. Therefore, the biofilms were not pre-grown at acidic conditions, which can be considered a limitation: there are multiple sites that are physiologically acidic and pH can have an effect on C. albicans morphology. On the other hand, acidic pH may favour fungal over bacterial growth as seen in Candida esophagitis, vulvovaginal candidosis and chronic wounds. Interestingly, the final metabolic activity and biomass of untreated control biofilms after 24 h exposure to pH 5.2 and 7.4 were comparable in the present study. A recent report suggested that composition of the medium has a major effect on biofilm architecture, expression profiles and antifungal susceptibility. In RPMI medium, the D-glucose concentration of 2.0 g/l is higher than the physiological concentration normally found in the human body.