In order to better study the biofilm formation process, image structure analyzer software was used to quantitatively analyze the biofilm formation process of Pseudomonas aeruginosa under different concentrations of glucose and allicin. Glucose solution (5.6, 7.0, 11.1, 16.7, 20 and 30 mmol/L) was added to the culture medium of non repetitive Pseudomonas aeruginosa and 6 groups were formed. Minimum inhibitory concentration of allicin to Pseudomonas aeruginosa PAO1 was determined by Tryptic Soy Broth micro dilution method. Single colony was inoculated into Tryptic Soy Broth according to garlic concentration (10; 128 μg/mL). Concomitantly, 3 groups were divided into 1, 3 and 7 d study. The groups of experimental materials were placed in confocal laser scanning microscope for observation and the obtained pictures were imported into the image structure analyzer software, through which the quantitative analysis of the biofilm structure was performed. As main findings we found that as glucose concentration increases, the thickness of Pseudomonas aeruginosa biofilm gradually increased. After glucose (30 mmol/L) treatment for 3 d, areal porosity decreased from 0.93±0.10 to 0.62±0.02 and the biofilm became thicker; textural entropy changed from 6.67±0.99 to 7.88±0.21, meaning an increase in biofilm heterogeneity and thickness; the average diffusion distance decreased from 2.44±0.28 to 1.72±0.36, the biofilm became thicker and the average diffusion distance between colonies also decreased. Under the intervention of allicin 10 μg/mL, the thickness of biomembrane was significantly reduced, the structure of biomembrane was sparse and the numbers of dead and living bacteria were significantly reduced. Under the action of allicin 128 μg/mL, the thickness of biomembrane was further reduced and the dead bacteria were clustered. After the action of allicin 128 μg/mL for 7 d, areal porosity increased from 0.68±0.10 to 0.92±0.02, the biomembrane became sparser; textural entropy increased from 6.67±0.93 changed to 5.52±0.548, the membrane heterogeneity decreased. Image structure analyzer software showed to be suitable for quantitative analysis of biofilm formation process. It is concluded that with the increase of glucose concentration, the maturation speed of Pseudomonas aeruginosa biofilm is accelerated, and the thickness of biofilm is gradually increased; allicin can destroy the self structure of Pseudomonas aeruginosa biofilm.