## 6 Conclusions  
In this paper, the protocol for a test we dubbed AntiBioVol is provided. Furthermore, the antibofilm activity of volatilized EOs against pathogenic biofilm was scrutinized in vitro using the AntiBioVol methodology. This method allowed a high number of repeats to be performed in a space- and cost-efficient manner. AntiBioVol has been compared to the standard Petri dish-based test. Not only do the results obtained by AntiBioVol display low standard deviations but also the use of resources is lower in comparison to standard tests. No expensive/advanced equipment is required for AntiBioVol performance. The application of AntiBioVol facilitates and improves the assessment of the effectiveness of antib biofilm volatile compounds.  
The following are available online at [[http://www.mdpi.com/2076-3417/10/20/7343/s1](http://www.mdpi.com/2076-3417/10/20/7343/s1)]([http://www.mdpi.com/2076-3417/10/20/7343/s1](http://www.mdpi.com/2076-3417/10/20/7343/s1)), Figure S1: GC-MS chromatograms of main EO ingredients. E-EO--eucalyptus oil; TT-EO--tea tree oil, T-EO--thyme oil. Results are presented as percentage content based on the peak area normalization. Figure S2: Photographic presentation of AntiBioVol test performance. Figure S3: Calibration curves for absorbance measurements at 490 nm (TTC assay) versus number of log10 colony-forming units (CFU)/mL [A] S. aureus, [B] _P. aeruginosa_. Figure S4: Distribution of 3 TT-EO's concentrations in a single test plate. TT-EO--tea tree oil; 25%, 50%, 100%: applied concentrations [\(\
icefrac{{v}}{{v}}\)] of TT-EO. Figure S5: Lack of PEG antimicrobial activity against _S. aureus_ in the AntiBioVol experimental setting. C+--positive control; 50%, 25% PEG--concentrations (\(\
icefrac{{v}}{{v}}\)) of polyethylene glycol. No significant difference (\(p<0.05\), K-W test, followed by [PERSON]'s analysis) between control and PEGs was detected with relation to S. aureus viability. Figure S6: Antibacterial activity of thyme essential oil liquid against S. aureus determined by the disc diffusion method (A) and inverted Petri plate method (B). Figure S7: Antibacterial activity of thyme essential oil liquid against _P. aeruginosa_ determined by the disc diffusion method (A) and inverted Petri plate method (B). Table S1: Composition of main ingredients of tested EOs. E-EO--eucalyptus essential oil; TT-EO--tea tree essential oil; T-EO--thyme essential oil; RI--retention index; RT--retention time. Table S2: Minimal Inhibitory Concentration (MIC) and Minimum Biofilm Eradication Concentration (MBEC) of eucalyptus, thyme, tea-tree EOs (E-EO, T-EO, and TT-EO, respectively) and ethanol (EtOH). Table S3: Antibiotic activity of volatile fractions of EOs and ethanol against _S. aureus_, _P. aeruginosa_, and _C. albicans_ biofilm. Table S4: Comparison of antibiofilm activity of three concentrations of TT-EO against _S. aureus_ biofilm.  
Conceptualization, M.B. (Malwina Brozyna) and A.J.; methodology, M.B. (Malwina Brozyna), A.J., K.F., A.Z.; validation, A.J., K.F.; formal analysis, M.B. ([PERSON]); investigation, M.B. ([PERSON]), A.J., K.F., A.Z., D.G., K.D., M.O.-W., B.D., P.M.; resources, K.F., M.B. ([PERSON]), M.B. ([PERSON]); data curation, M.B. ([PERSON]); writing--original draft preparation, M.B. ([PERSON]), A.J.; writing--review and editing, K.F., A.J., A.Z., M.B. ([PERSON]); visualization, M.B. ([PERSON]), A.Z.; supervision, K.F., A.J.; project administration, A.J., K.F.; funding acquisition, K.F., A.J., M.B. ([PERSON]). All authors have read and agreed to the published version of the manuscript.  
This study was supported by the National Science Center (Grant No. 2017/27/B/NZ6/02103) and STMD 230.20.126 funding from Wroclaw Medical University.  
The authors declare no conflict of interest.